A gradient has been designed to yield two subfractions of plasma membrane vesicles from rat myometrium, a low buoyant density (8-24% sucrose) fraction N1 richer in 5'-nucleotidase and a higher buoyant density (24-30% sucrose) fraction N2, instead of a previously described fraction F1. Both N1 and N2 had very low activities of NADPH-cytochrome c reductase and succinate-cytochrome c reductase. Electron micrographs of thin sections of N1 showed clear vesicles, whereas N2 consisted of vesicles with electron-dense bodies attached to them. These plasma membrane vesicles can actively take up Ca. The active uptake of Ca was potentiated by oxalate and phosphate and abolished by the Ca ionophore A23187. Dilution of actively loaded vesicles in isotonic media containing EGTA led to loss of a small proportion of the stored Ca instantaneously and the remainder more slowly in a biphasic manner. Dilution in hypotonic media with EGTA led to a release of a much larger proportion of the accumulated Ca. A23187 at high concentrations (10 microM) caused a release of all the sequestered Ca whether the active Ca uptake had been carried out in the presence or in the absence of oxalate. A23187, 0.5 microM, released all the sequestered Ca from the vesicles that were actively loaded in the absence of oxalate, but only 37% when the vesicles were actively loaded with Ca in the presence of oxalate. Comparison of the composite plasma membrane fraction F1 (8-30% sucrose) and the subfractions N1 and N2 showed that they had different capacities for Ca uptake in the presence and absence of ATP. An attempt has been made to analyze the active Ca-uptake data in terms of various Ca pools.