A high-affinity calcium-stimulated ATPase (Ca-ATPase) was identified in a plasma membrane subcellular fraction from human liver. The subcellular distribution of the enzyme corresponds to that of classical plasma membrane markers, and its kinetic parameters are identical to those of the Ca-ATPase identified in rat liver plasma membranes in that: (i) calcium activation of ATPase activity followed a cooperative mechanism (Hill number = 1.2), half-maximal activation occurring at 4.5 ± 2.0 nm free calcium; (ii) K0.5 of the enzyme for ATP was 54 ± 5 μM, and kinetic studies indicated that two interacting sites were involved; (iii) micromolar concentrations of MgCl2 had an inhibitory effect on enzyme activity, and (iv) the enzyme did not require potassium for activation by calcium and was not sensitive to ouabain or oligomycin. These properties are characteristic of Ca-ATPases coupled to calcium pumps in plasma membranes. The effects of the local anesthetic, tetracaine, and three calcium antagonists bepridil, verapamil, nifedipine, were investigated. Bepridil at 50 μM caused 75% inhibition of Ca-ATPase activity by decreasing the maximal velocity of the enzyme reaction; its action was immediate and reversible. Verapamil (0.5 μM) also induced 75% inhibition of the Ca-ATPase, but its affinity for the system was a third of that of bepridil. The enzyme was much less sensitive to nifedipine and tetracaine. The method described in the present paper for measuring Ca-ATPase activity in small human liver biopsies will permit the study of this enzyme during the course of hepatic disorders.