A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone. Copyright © 2005 John Wiley & Sons, Ltd.166411414Bringmann, G., Saeb, W., Assi, L.A., François, G., Narayanan, A.S.S., Peters, E.-M., Betulinic acid: Isolation from Triphyophyllum peltatum and Ancistrocladus heyneanus, anti-malarial activity and crystal structure of the benzyl ester (1997) Planta Med., 63, pp. 255-257Budzikiewicz, H., Wilson, J.M., Djerassi, C., Mass spectrometry in structural and stereochemical problems. XXXII. Pentacyclic triterpenes (1963) J. Am. Chem. Soc., 85, pp. 3688-3699Conway, W.D., (1990) Countercurrent Chromatography: Apparatus, Theory and Applications, , VCH-Wiley: New YorkDräger, D., Neubert, R., Galgon, T., Wohlrab, W., Verfahren zur Extraktion von Betulinsäure aus der Borke von Platanus acerifolia unter Nutzung mitterpolarer Lösungsmittel (2001), Ger Offen DE 19,713,768Fujioka, T., Kashiwada, Y., Kilkuskie, R.E., Cosentino, L.M., Ballas, L.M., Jiang, J.B., Janzen, W.P., Lee, K.-H., Anti-AIDS agents, 11. Betulinic acid and platanic acid as anti-HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally related triterpenoids (1994) J. Nat. Prod., 57, pp. 243-247Hayek, E.W.H., Jordis, U., Moche, W., Sauter, F., A bicentennial of betulin (1989) Phytochemistry, 28, pp. 2229-2242Hostettmann, K., Marston, A., Countercurrent chromatography in the preparative separation of plant-derived natural products (2001) J. Liq. Chrom. Relat. Technol., 24, pp. 1711-1721Ito, Y., Oka, H., Kitazume, E., Bhatnagar, M., Lee, Y.W., Improved high-speed countercurrent chromatograph with three multilayer coils connected in series. III. Evaluation of semianalytical column with various biological samples and inorganic elements (1990) J. Liq. Chromatogr., 3, pp. 2329-2349Junges, M.J., Fernandes, J.B., Rodrigues-Filho, E., Vieira, P.C., Silva, M.F.G.F., Potenciais anti-AIDS isolados de Eugenia florida (1994) XIII Simpósio De Plantas Medicinais Do Brasil, 1, p. 135. , Anais do CongressoKim, D.S.H.L., Chen, Z., Nguyen, V.T., Pezzuto, J.M., A concise semi-synthetic approach to betulinic acid from betulin (1997) Synth. Commun., 29, pp. 1607-1612Mabberley, D.J., (1998) The Plant Book - A Portable Dictionary of the Vascular Plants, , (2nd edn). Cambridge University Press: LondonMarston, A., Hostettmann, K., Counter-current chromatography as a preparative tool - Applications and perspectives (1994) J. Chromatogr. A, 658, pp. 315-341Pisha, E., Chai, H., Lee, I.-S., Chagwedera, T.E., Farnsworth, N.R., Cordell, G.A., Beecher, C.W.W., Pezzuto, J.M., Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis (1995) Nature Med., 1, pp. 1046-1051Sun, I.-C., Shen, J.-K., Wang, H.-K., Cosentino, L.M., Lee, K.-H., Anti-AIDS agents. 32. Synthesis and anti-AIDS activity of betulin derivatives (1998) Bioorg. Med. Chem. Lett., 8, pp. 1267-1272Wagner, H., Bladt, S., (1996) Plant Drug Analysis, , 2nd edn. Springer: Berlin