Thyroid hormone (10(-11) to 10(-10) M) stimulates plasma membrane Ca2+-ATPase activity in vitro in various tissues, including the human red cell (RBC), by a calmodulin-requiring mechanism. Bepridil and cetiedil are Ca2+ antagonists with an intracellular (calmodulin-antagonist) site of action, as well as an effect on the calcium channel in excitable tissues. We have studied the actions of bepridil and cetiedil on Ca2+-ATPase in a channel-free membrane (RBC) to determine effectiveness of these agents as inhibitors of thyroid hormone action on the enzyme. Dose-response studies showed that thyroid hormone stimulation of Ca2+-ATPase activity in vitro was significantly inhibited by as little as 2 x 10(-5) M bepridil and cetiedil. IC50 values of bepridil and cetiedil for thyroid hormone response of the enzyme were 5 x 10(-5) and 2 x 10(-5) M, respectively, whereas IC50s of these agents for enzyme activity in the absence of thyroid hormone were both 10(-4) M. Progressive addition of purified rat testis calmodulin in vitro (10-150 ng calmodulin/mg membrane protein) restored hormone responsiveness in the presence of bepridil and cetiedil. Binding of labeled thyroid hormone by RBC membranes was unaffected by bepridil and cetiedil (up to 2 x 10(-4) M). Thus, bepridil and cetiedil are Ca2+ antagonists that reversibly inhibit thyroid hormone action on human RBC Ca2+-ATPase by a calmodulin-dependent mechanism. Thyroid hormone effect on Ca2+-ATPase is more susceptible to bepridil and cetiedil inhibition than is basal enzyme activity.