In resting smooth muscle, a variety of Ca2+ extrusion processes offset the inward Ca2+ leak. Biochemical studies suggest that the plasmalemmal Ca2+ pump dominates this process; however, this contention could not be proven without a reliable estimate of the inward Ca2+ leak that must be opposed by active transport. Recent studies using dispersed cells from the toad stomach provided such an estimate; thus we examined the capacity of the plasmalemmal Ca2+ pump in this tissue. Membranes were prepared using nitrogen cavitation, high-salt extraction, and flotation on discontinuous sucrose gradients. These membrane vesicles were enriched 16- to 24-fold for plasma membrane markers and exhibited an ATP-dependent uptake of 45Ca that was insensitive to azide or oxalate but sensitive to orthovanadate inhibition and calmodulin stimulation. 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by caffeine, inositol 1,4,5-trisphosphate, or GTP. Uptake exhibited a high affinity for Ca2+ (Km 0.2 microM) and a high-transport capacity, producing greater than 12,000-fold gradient for Ca2+ and a transmembrane flux rate greater than that observed in resting smooth muscle cells. Thus this enzyme is capable of maintaining steady-state Ca2+ levels in smooth muscle.