ATP-dependent Ca uptake by rat myometrium plasma membrane fraction occurs only in the presence of magnesium. Studies of the membrane protein-concentration dependence of the ATP-dependent Ca uptake showed that at 33 micrograms protein/ml the uptake increased linearly with time up to 2 min and deviated from linearity at longer times. Studies of the Ca2+ concentration dependence of initial Ca-uptake velocity (reaction time 2 min) of the uptake reaction showed that 1) ATP-dependent Ca uptake increased with increasing Ca2+ concentration from 0.1 to 1 microM, and a further increase in Ca2+ concentration above 1 microM did not enhance the uptake further and 2) the Ca-uptake dependence on Ca2+ concentration was nonhyperbolic with a Hill coefficient of 1.3–1.7 and half saturation occurring at 0.4–0.5 microM Ca2+. These data suggest that the ATP-dependent Ca movement by rat myometrium plasma membranes may play a very important role in smooth muscle relaxation. Physiological implications of the nonhyperbolic kinetics are also discussed.