To explore further the mechanisms that regulate the Na+/H+ antiport in human platelets, we examined the effect of Na+ pump inhibition by ouabain and K+ removal from the extracellular medium on parameters of this transport system. Treatment with ouabain resulted in increased cytosolic free Ca2+ and Na+, coupled with an alkaline shift in the cytosolic pH set point for the Na+/H+ antiport. Inhibition of the Na+ pump by the removal of K+ from the medium increased the cytosolic Na+ but not the cytosolic Ca2+; yet this treatment also produced a substantial alkaline shift in the cytosolic pH set point for the Na+/H+ antiport. This effect appeared to relate to a decline in cellular volume and it was attenuated by the Na(+)-K(+)-2 Cl- cotransport inhibitor, bumetanide. These findings indicate: (a) a link between the Na+ pump and the Na+/H+ antiport, mediated by the Na+/Ca2+ exchange and the cytosolic free Ca2+, and (b) a link between the Na+/H+ antiport and the Na(+)-K(+)-2Cl- cotransport through cellular volume.