Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (NCX1.1) accumulated Cd2+ after a lag period of several tens of seconds. The lag period reflects the progressive allosteric activation of exchange activity by Cd2+ as it accumulates within the cytosol. The lag period was greatly reduced in cells expressing a mutant exchanger, (241-680), that does not require allosteric activation by Ca2+ for activity. Non-transfected cells did not show Cd2+ uptake under the same conditions. In cells expressing NCX1.1, the lag period was nearly abolished following an elevation of the cytosolic Ca2+ concentration. Cytosolic Cd2+ concentrations estimated at 0.5-2 pM markedly stimulated the subsequent uptake of Ca2+ by Na+/Ca2+ exchange. Outward exchange currents in membrane patches from Xenopus oocytes expressing the canine NCX1.1 were rapidly and reversibly stimulated by 3 pM Cd2+ applied at the cytosolic membrane surface. Exchange currents activated by 3 pM Cd2+ were 40% smaller than currents activated by 1 µM cytosolic Ca2+. Current amplitudes declined by 30% and the rate of current development fell sharply upon repetitive applications of Na+ in the presence of 3 pM Cd2+. Cd2+ mimicked the anomalous inhibitory effects of Ca2+ on outward exchange currents generated by the Drosophila exchanger CALX1.1. We conclude that the regulatory sites responsible for allosteric Ca2+ activation bind Cd2+ with high affinity and that Cd2+ mimics the regulatory effects of Ca2+ at concentrations 5 orders of magnitude lower than Ca2+.