Three distinct mammalian Na+/Ca2+ exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken a detailed functional comparison of these three exchangers. Each exchanger was stably expressed at high levels in the plasma membranes of BHK cells. Na+/Ca2+ exchange activity was assessed using three different complementary techniques: Na+ gradient-dependent 45Ca2+ uptake into intact cells, Na+ gradient-dependent 45Ca2+ uptake into membrane vesicles isolated from the transfected cells, and exchange currents measured using giant patches of excised cell membrane. Apparent affinities for the transported ions Na+ and Ca2+ were markedly similar for the three exchangers at both membrane surfaces. Likewise, generally similar responses to changes in pH, chymotrypsin treatment, and application of various inhibitors were obtained. Depletion of cellular ATP inhibited NCX1 and NCX2 but did not affect the activity of NCX3. Exchange activities of NCX1 and NCX3 were modestly increased by agents that activate protein kinases A and C. All exchangers were regulated by intracellular Ca2+. NCX1-induced exchange currents were especially large in excised patches and, like the native myocardial exchanger, were stimulated by ATP. Results may be influenced by our choice of expression system and specific splice variants, but, overall, the three exchangers appear to have very similar properties.