Shaker genes encode voltage-gated potassium channels (Kv). We have shown previously that genes from Shaker subfamilies Kv1.1, 1.2, 1.4 are expressed in rabbit kidney. Recent functional and molecular evidence indicate that the predominant potassium conductance of the kidney medullary cell line GRB-PAP1 is composed of Shaker-like potassium channels. We now report the molecular cloning and functional expression of a new Shaker-related voltage-gated potassium channel, rabKv1.3, that is expressed in rabbit brain and kidney medulla. The protein, predicted to be 513 amino acids long, is most closely related to the Kv1.3 family although it differs significantly from other members of that family at the amino terminus. In Xenopus oocytes, rabKv1.3 cRNA expresses a voltage activated K current with kinetic characteristics similar to other members of the Kv1.3 family. However, unlike previously described Shaker channels, it is sensitive to glibenclamide and its single channel conductance saturates. This is the first report of the functional expression of a voltage-gated K channel clone expressed in kidney. We conclude that rabKv1.3 is a novel member of the Shaker superfamily that may play an important role in renal potassium transport.