A membrane-associated, Ca(2+)-activated, Mg(2+)-dependent ATPase activity has been identified in the mouse chorioallantoic placenta. The enzyme activity is expressed and increases as a function of gestation. Biochemical characterization shows that the enzyme is highly specific for Ca2+ and nucleotide triphosphates, with a Km of 0.97 mM [Ca2+] and a Vmax of 1.05 nmol Pi released mg-1 placental protein min-1. The mouse placental Ca(2+)-ATPase activity has a pI of approximately 6.8, and corresponds to two apparent Mr values of 118 and 150 x 10(3), based on Ferguson analysis of non-denaturing electrophoretograms. Enzyme activity is inhibited by phenothiazin (suggesting a calmodulin dependence), vanadate, erythrosin B and quercetin, but not by ouabain or levamisole. Enzyme cytohistochemistry revealed that the Ca(2+)-ATPase is localized to polyploid trophoblastic cells of the mouse inner placenta. These results suggest that the enzyme may be a functional component of transplacental calcium transport during mouse embryonic development.