The metabolism, flagellar beating, and acrosome reaction of spermatozoa are regulated by ion flux across the plasma membrane. As is true of most cells, swimming sperm maintain intracellular Ca(2+) concentrations at submicromolar levels. Here we describe a K(+)-dependent Na(+)/Ca(2+) exchanger (suNCKX) from sea urchin sperm. The suNCKX is phylogenetically related to other NCKXs, which use high relative intracellular K(+), and high relative extracellular Na(+), to couple the efflux of 1 Ca(2+) and 1 K(+) to the influx of 4 Na(+). The 652-aa suNCKX shares structural topology with other NCKX proteins, and has two protein kinase A sites and a His-rich region in its cytoplasmic loop. The suNCKX is encoded by a single gene, which is highly expressed in testes. The suNCKX activity of whole sperm shows Na(+) and K(+) dependence, and like other NCKXs can run in reverse exchange mode. An inhibitor blocks the suNCKX activity and sperm motility. suNCKX localizes to the plasma membrane over the sperm flagellum. The suNCKX may play a major role in keeping Ca(2+) low in swimming sperm.