Phosphatidylserine (PtdSer)-liposomes when incubated with synaptosomal plasma membranes (SPM) of dog brain, evoked a significant increase (approx 80%) of the Ca2+-stimulated ATPase activity with maximal effect achieved at around 0.7 mumol PtdSer/mg SPM protein. Higher concentrations of PtdSer led to inhibition of the enzyme activity with respect to the maximal percentage of stimulation. Treatment of SPM with EGTA, to minimize the presence of bound cytoplasmic activator calmodulin, resulted in a mixed mechanism of inhibition of the enzyme activity (Vmax was decreased and Km increased) as estimated by Lineweaver-Burk plots. Addition of exogenous calmodulin resulted in an increase of Vmax and in a restoration of Km to control value. Ca2+-stimulated ATPase activity, in EGTA-treated SPM, showed the same figure of changes at different concentrations of PtdSer-liposomes as those of the control, but the turning point was now located at higher PtdSer concentrations. The results suggest that Ca2+-stimulated ATPase activity of SPM is modulated by PtdSer and that calmodulin participates in these interactions, probably, by regulating the contact between the enzyme and Ca2+ ions.