In zebrafish (Danio rerio), six distinct Na+-K+-ATPase (NKA) alpha1-subunit genes have been identified, and four of them, zatp1a1a.1, zatp1a1a.2, zatp1a1a.4, and zatp1a1a.5, are expressed in embryonic skin where different types of ionocytes appear. The present study attempted to test a hypothesis of whether these NKA alpha1 paralogues are specifically expressed and function in respective ionocytes. Double fluorescence in situ hybridization analysis demonstrated the specific expression of zatp1a1a.1, zatp1a1a.2, and zatp1a1a.5 in NKA-rich (NaR) cells, Na+-Cl- cotransporter (NCC)-expressing cells, and H+-ATPase-rich (HR) cells, respectively, based on the colocalization of the three NKA alpha1 genes with marker genes of the respective ionocytes (epithelial Ca2+ channel in NaR cells; NCC in NCC cells; and H+-ATPase and Na+/H+ exchanger 3b in HR cells). The mRNA expression (by real-time PCR) of zatp1a1a.1, zatp1a1a.2, and zatp1a1a.5 were, respectively, upregulated by low-Ca2+, low-Cl-, and low-Na+ freshwater, which had previously been reported to stimulate uptake functions of Ca2+, Cl-, and Na+. However, zatp1a1a.4 was not colocalized with any of the three types of ionocytes, nor did its mRNA respond to the ambient ions examined. Taken together, zATP1a1a.1, zATP1a1a.2, and zATP1a1a.5 may provide driving force for Na+-coupled cotransporter activity specifically in NaR, NCC, and HR cells, respectively.