Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phospho-amino-acids and P-32-casein under physiologically appropriate conditions. PNPP was hydrolysed at a rate of 80 nmol.mg(-1).h(-1) in the presence of 5 mM MgCl2, pH 7.2 at 30 degrees C. In the absence of Mg2+ the activity was reduced 40% and we call this basal activity. At saturating concentration of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mM MgCl2. Ca2+ had no effect on the basal activity, could not substitute Mg2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg2+ (I-50 = 0.43 mM). In the absence of Mg2+ (basal activity) the stimulating half concentration (S-0.5) for PNPP was 1.57 mM, while at saturating MgCl2 concentrations the corresponding S-0.5 for PNPP for Mg2+-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mM. The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amastigotes were also able to hydrolyse phosphoserine, phosphothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolyse P-32-casein phosphorylated on serine and threonine residues but only in the presence of MgCl2. These results indicate that in the amastigote form of T.: cruzi there are at least two ectophosphatase activities, one of which is Mg2+ dependent and can dephosphorylate phospho-aminoacids and phosphoproteins under physiological conditions.5411977984