Abstract We have identified and characterized cDNAs encoding a novel zebrafish Na,K-ATPase α subunit. The full-length cDNA encodes a 1023-amino-acid-long peptide which shows greatest homology to zebrafish α1 polypeptides. Radiation hybrid mapping localized the new gene ( atp1a1a.5) to linkage group 1 in close proximity to the previously identified cluster of Na,K-ATPase α1 genes. The expression of atp1a1a.5 in zebrafish embryos was analyzed using whole-mount in situ hybridization. From mid-somitogenesis through 48 h post fertilization (hpf), atp1a1a.5 transcripts were detected in the pronephric duct, ear, and mucous cells. This expression pattern continues through 108 hpf, when high levels of expression were also detected in the intestinal bulb.