It has been reported that the luteal function may be regulated by the intracellular calcium in luteal cells (Higuchi et al, 1976; Dorflinger et at, 1984; Gore and Behrman, 1984) which is adjusted partially by Ca⁺⁺-ATPase activities in luteal cell membranes (Verma and Pennistion, 1981). However, the physicochemical and kinetic properties of Ca⁺⁺-ATPase in luteal membranes were not fully characterized. This study was, therefore, undertaken to partially characterize the physicochemical and kinetic properties of Ca⁺⁺-ATPase system in luteal membranes and microsomal fractions, known as an one of the major Ca⁺⁺ storge sites (Moore and Pastan, 1978), from the highly luteinized ovary Highly luteinized ovaries were obtained from PMSG-hCG injected immautre female rats. Light membrane and heavy membrane fractions and microsomal fractions were prepared by the differential and discontinuous sucrose density gradient centrifugation method desribed by Bramley and Ryan (1980). Light membrane and heavy membrane fractions and microsomal fractions from highly luteinized ovaries are composed of the two different kinds of Ca⁺⁺-ATPase system. One is the high affinity Ca⁺⁺-ATPase which is activated in low Ca⁺⁺ concentration (Km, 10-30 nM), the other is low affinity Ca⁺⁺-ATPase activated in higher Ca⁺⁺ concentration (K_½, 40 μM). At certain Ca⁺⁺ concentrations, activities of high and low affinity Ca⁺⁺-ATPase are the highest in light membrane fractions and are the lowest in microsomal fractions. It appeares that high affinity Ca⁺⁺-ATPase system have 2 binding sites for ATP (Hill s coefficient; around 2 in all membrane fractions measured) and the positive cooperativity of ATP bindings obviously existed in each membrane fractions. The optimum pH for high affinity Ca⁺⁺-ATPase activation is around S in all membrane fractions measured. The lipid phase transition temperature measured by Arrhenius plots of high affinity Ca⁺⁺-ATPase activity is around 25℃. The activation energies of high affinity Ca⁺⁺-ATPase below the transition temperature are similar in each membrane fractions, but at the above transition temperature, it is the hightest in heavy membrane fractions and the lowest in microsomal fractions. According to the above results, it is suggested that intracellular Ca⁺⁺ level, which may regulate the luteal function, may be adjusted primarily by the high affinity Ca⁺⁺-ATPase system activated in intracellular Ca⁺⁺ concentration range (below 0.1 μM).