Sphingomyelins (SM) and phosphatidylcholines (PC) are major lipid classes in the external plasma membrane leaflet of mammalian cells. A preferential interaction between SM and cholesterol (Cho) in both cell and model membranes has been proposed as central for the formation of Cho- and SM-rich domains in membranes. In this context, the relevance of the SM hydrophobic moiety on its interaction with Cho for domain stabilization has been investigated by our group (1-2). We report here on the effects of sphingomyelin structure on the orientational and conformational properties of monolayers of pure lipids and of two ternary lipid mixtures (DOPC/16:0SM/Cho and DOPC/24:1SM/Cho), which are relevant as mammalian cell membrane models. We investigated interchain interactions, hydrogen bonding, conformational and structural properties using in situ polarization modulated infrared reflection absorption spectroscopy (PM-IRRAS). Our results indicate that the particular properties conferred on sphingolipids by unsaturation have profound implications on membrane organization.Finally, we also explored the orientational and conformational changes in lipid monolayers of DOPC/16:0SM/Cho 2:1:1 after the adsorption/insertion of the active toxin HlyA and its unacylated nonhemolytic precursor ProHlyA, so as to complement our knowledge on the action mechanism of both proteins.Instituto de Investigaciones Bioquímicas de La PlataComisión de Investigaciones Científicas de la provincia de Buenos AiresInstituto de Investigaciones Fisicoquímicas Teóricas y AplicadasCentro de Investigación de Proteínas Vegetales