Effects of phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylcholine (PC) on high and low affinity 45Ca uptake (ATP present) and binding (ATP absent) in microsomes prepared from canine aortic media-intima were examined. When high affinity Ca++ uptake predominated (in 0.03 mM Ca++), PS and PI (but not PC) increased 45Ca binding but not 45Ca uptake. Only PI increased binding and uptake when low affinity Ca++ uptake predominated (in 3.0 mM Ca++). Oxalate-potentiated Ca++ uptake was inhibited by only PS and PI. PS and PI induced a reuptake of 45Ca that was blocked or reversed by EGTA (0.1 mM) or neomycin (0.14 mM). The Ca++ ionophore A23187 (10(-6) M) decreased high affinity 45Ca uptake to a level similar to that of high affinity 45Ca binding and, when added during 45Ca washout, increased 45Ca loss. Net negatively charged exogenous PS or PI may enter the microsomal membranes and provide additional high affinity Ca++ binding sites. The actions of PS and PI differ qualitatively from those of the Ca++ ionophore A23187.