We compared the properties of three mammalian Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, by analyzing the effects of Ni2+ and other cations as well as the recently identified inhibitor isothiourea derivatives on intracellular Na+-dependent 45Ca2+ uptake into CCL-39 (Dede) fibroblasts stably expressing each isoform. All these NCX isoforms had similar affinities for the extracellular transport substrates Ca2+ and Na+. Ni2+ inhibited 45Ca2+ uptake by competing with Ca2+ for the external transport site, with 10-fold less affinity in NCX3 than in NCX1 or NCX2. Ni2+ and Co2+ were most efficient in such discrimination of NCX isoforms, although their inhibitory potencies were less than those of La3+ and Cd2+. The monovalent cation Li+ stimulated 45Ca2+ uptake rate by all NCX isoforms similarly with low affinity, although the extent of stimulation was somewhat smaller in NCX1. On the other hand, the isothiourea derivative KB-R7943 was threefold more inhibitory to NCX3 than to NCX1 or NCX2. Thus distinct differences in the kinetic and pharmacological properties were detected between NCX3 and the other two isoforms.