The retinal Na+:Ca2+, K+exchanger cDNA was transiently expressed in human embryonic kidney (HEK 293) cells by transfection with plasmid DNA. The correct targeting of the expressed protein to the plasma membrane was confirmed by immunocytochemistry. The reverse exchange offrent (Ca2+ imported per Na+ extruded) was measured in whole-cell voltage-clamp experiments after intracellular perfusion with Na+ (Na+i, 128 mM) and extracellular perfusion with Ca2+ (Ca2o+, 1 mM) and Ko+ (20 mM). As expected, the exchange current was suppressed by removing Ca2o+. Surprisingly, however, it was also abolished by increasing Na+o to almost abolish the Na+ gradient, and it was almost unaffected by the removal of Ko+. Apparently, then, at variance with the exchanger in the rod outer segment, the retinal exchanger expressed in 293 cells acts essentially as a Na+:Ca2+ exchanger and does not require K+ for its electrogenic activity.