The Na-K-Cl cotransporter isoform 1 (NKCC1) has been isolated from several species, including Squalus acanthias. A second kidney-specific isoform (NKCC2) has been cloned mainly from higher vertebrates. Here, we have isolated the S. acanthias NKCC2 and found that it is produced in at least four spliced variants (saNKCC2A, saNKCC2F, saNKCC2AF, and saNKCC2AFno8) of ∼1,090 residues. Expression of these transcripts in Xenopus laevis oocytes revealed that only the A and F variants are functional and that they are more active after incubation in low-Cl or hyperosmolar media. Rates of activation after exposure to these media were exceptionally rapid, demonstrating for the first time that the NKCC2 itself represents an important site of regulation by Cl and that extracellular domains are involved. Another remarkable finding in this study was the failure to identify NKCC2B, a variant found in the kidney of higher vertebrates and expressed specifically in macula densa cells. This result, in conjunction with the fact that the shark kidney lacks a well-developed juxtaglomerular apparatus, suggests that the B exon evolved as a result of selective pressure (presumably by exon duplication) and that a restricted relationship exists between NKCC2B and macula densa.