We used DAMI human megakaryocytic leukemia cells to study transmembrane ion currents activated through the G-protein-coupled thrombin receptor pathway. When the cells were stimulated by thrombin receptor-activating peptide, an increase in cytosolic Ca2+ ([Ca2+]i) developed as predicted by the known effect that thrombin exerts in the platelet. We then monitored the membrane potentials of individual DAMI cells during this response and observed complex, triphasic changes that could not be accounted for by Ca2+ fluxes alone. These consisted of rapid hyperpolarization, followed by depolarization to values more positive than the resting potential and then by slow repolarization. For the purpose of this study, we focused on the hyperpolarizing current that developed immediately after thrombin receptor activation. This proved to be composed of (1) a Ca(2+)-independent, outwardly rectifying Cl- current and (2) a strongly hyperpolarizing, inwardly rectifying, Ba(2+)- sensitive K+ current that required an increase of [Ca2+]i for activation. By analogy with their functions in other cell systems, it is logical to conclude that these prominent K+ and Cl- conductances may serve to regulate the complex volume changes that accompany thrombin receptor activation and/or to increase the electromotive drive that supports Ca2+ influx under these conditions through hyperpolarization of the cell membrane.