ATP-sensitive K⁺ (K_ATP) channels play a crucial role in coupling cellular metabolism to membrane potential. In addition to the orthologs corresponding to Kir6.1 and Kir6.2 of mammals, we have identified a novel member, designated Kir6.3 (zKir6.3), of the inward rectifier K⁺ channel subfamily Kir6.x in zebrafish. Zebrafish Kir6.3 is a protein of 432 amino acids that shares 66% identity with mammalian Kir6.2 but differs considerably from mammalian Kir6.1 and Kir6.2 in the C-terminus, which contain an RKR motif, an endoplasmic reticulum (ER)-retention signal. Single-channel recordings of reconstituted channels show that zebrafish Kir6.3 requires the sulfonylurea receptor 1 (SUR1) subunit to produce K_ATP channel currents with single channel conductance of 57.5 pS. Confocal microscopic analysis shows that zebrafish Kir6.3 requires the SUR1 subunit for its trafficking to the plasma membrane. Analyses of chimeric protein between human Kir6.2 and zebrafish Kir6.3 and a C-terminal deletion of zebrafish Kir6.3 indicate that interaction between the C-terminus of zebrafish Kir6.3 and SUR1 is critical for both channel activity and trafficking to the plasma membrane. We also identified zebrafish orthologs corresponding to mammalian SUR1 (zSUR1) and SUR2 (zSUR2) by the genomic database. Both Kir6.3 and SUR1 are expressed in embryonic brain of zebrafish, as assessed by whole-mount in situ hybridization. These data indicate that Kir6.3 and SUR1 form functional K_ATP channels at the plasma membrane in zebrafish through a mechanism independent from ER-retention by the RKR motif.