Ca2+ influx via Na+/Ca2+ exchange was examined using fura-2 fluorescence techniques in human platelets loaded internally with Na+ by pretreatment with ouabain. In 140 mM LiCl, Ca2+ influx via the exchanger was completely dependent upon the presence of extracellular K+ (Km approximately 1 mM). In 140 mM N-methyl-D-glucamine (NMDG), Ca2+ influx was stimulated by K+ but was not absolutely dependent upon it; the inclusion of 20 mM NaCl in the NMDG medium restored the K+ dependence of Ca2+ influx. Stimulation of Ca2+ influx by K+ was confirmed by 45Ca2+ flux studies. 86Rb+ fluxes were measured to determine if Ca2+ and Rb+ were co-transported by the exchanger. The presence of extracellular Ca2+ stimulated 86Rb+ influx in ouabain-treated platelets in 160 mM LiCl or NMDG. The Rb+/Ca2+ influx ratio was 0.42 +/- 0.04 (n = 3) at [Rb+] = 0.2 mM and 0.89 at [Rb+] = 1.3 mM. Neither K(+)-dependent Ca2+ influx nor Ca(2+)-dependent Rb+ influx was observed in 160 mM NaCl or in platelets that had not been pretreated with ouabain, indicating that these fluxes resulted from Na+/Ca2+ exchange activity. Na(+)-dependent Ca2+ efflux was also shown to be dependent upon the presence of internal K+. These properties are similar to those of the Na+/Ca(2+)-K+ exchanger found in retinal rods, as distinct from the more widely distributed cardiac-type exchanger.