Inside-out vesicles prepared from human red blood cells took up Ca2+ by an active transport process. Membranes from the same red blood cells displayed Ca2+-activated, Mg2+-dependent adenosine triphosphatase activity. Both the initial rate of Ca2+ transport and the (Ca2++Mg2+)-adenosine triphosphatase activity were increased approximately twofold by the calcium binding protein, calmodulin. Activities in the absence of added calmodulin were termed basal activities. Calmodulin-activated Ca2+ transport and adenosine triphosphatase activities could be antagonized in a relatively selective fashion by the phenothiazine tranquilizer drug, trifluoperazine. High concentrations of trifluoperazine also inhibited basal Ca2+ transport and adenosine triphosphatase activity. By contrast, calmodulin binding protein from beef brain selectively antagonized the effect of calmodulin on Ca2+ transport with no inhibition of basal activity. Ruthenium red antagonized calmodulin-activated and basal activity with equal potency. The results demonstrate that although phenothiazines can act as relatively selective antagonists of calmodulin-induced effects, other effects are possible and cannot be ignored. Calmodulin-binding protein may be a useful tool in the analysis of calmodulin functions. Ruthenium red probably interacts with Ca2+ pump adenosine triphosphatase at a site not related to calmodulin.