The accumulation of Ca2+ and factors influencing Ca2+ incorporation in vascular smooth muscle were examined in a microsomal fraction prepared from canine aortae. The accumulation of Ca2+ required the presence of ATP and increased over time. The incorporation of 45Ca into the microsomal preparation was stimulated both in the presence of oxalate and when the Ca2+ concentration of the bathing media was elevated from 20 .mu.M-40 .mu.M. Ca uptake (with oxalate) was temperature-dependent; uptake was unaffected by azide. Both La3+ and phosphatidyl serine (PS) inhibited ATP-dependent uptake in a concentration-dependent manner. In the presence of ATP, Ca2+ binding (without oxalate) was slightly inhibited by La3+ PS had no effect under these conditions. When ATP was removed from the incubation medium, the presence of PS stimulated the uptake and binding of 45Ca; La3+ decreased uptake and binding. La3+ binds on the surface of the vesicles and, in this manner, decreases available Ca2+ sites; PS appears to increase the number of Ca2+ sites by either entering the membrane or adsorbing on it.