The transport stoichiometry is an essential property of antiporter and symporter transport proteins. In this study, we determined the transport stoichiometry of the retinal cone potassium-dependent Na/Ca exchanger (NCKX) expressed in sodium-loaded cultured insect cells. The Na/Ca and Rb/Ca coupling ratios were obtained by direct measurements of the levels of (86)Rb and (45)Ca uptake and sodium release associated with reverse Na/Ca exchange. Rb/Ca coupling ratios of 0.98 [standard deviation (SD) of 0.12, 15 observations] and 0.92 (SD of 0.12, 13 observations) were obtained for the chicken and human retinal cone NCKX, respectively. Na/Ca coupling ratios of 4.11 (SD of 0.24, 10 observations) and 3.98 (SD of 0.34, 15 observations) were obtained for the chicken and human retinal cone NCKX, respectively, whereas a lower average coupling ratio of 3.11 (SD of 0.34, 10 observations) was obtained with cells expressing the bovine Na/Ca exchanger (NCX1). These results are consistent with a 4Na/1Ca + 1K stoichiometry for retinal cone NCKX. High Five cells expressing full-length dolphin rod NCKX, Caenorhabditis elegans NCKX, or bovine rod NCKX from which the two large hydrophilic loops were removed all showed a significant calcium-dependent (86)Rb uptake, whereas no calcium-dependent (86)Rb uptake was observed in cells expressing bovine NCX1. The calcium dependence of (45)Ca uptake yielded values between 1 and 2.5 microM for the external calcium dissociation constant of the different NCKX proteins studied here.