We investigated the relationship between nitric oxide (NO) and Na(+),K(+)-ATPase (NKA) in the gill of anadromous Atlantic salmon. Cells containing NO-producing enzymes were revealed by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, which can be used as an indicator of NOS activity, i.e. NO production. Antibodies against the two constitutive NOS isoforms, neuronal and endothelial NOS, both produced immunoreactivity restricted to large cells at the base and along the secondary lamellae. NADPHd-positive cells showed a corresponding distribution. Antibodies against the inducible NOS isoform only labeled small cells located deep in the filament. Using in situ hybridization and NKA immunoreactivity, cells expressing Na(+),K(+)-ATPase alpha-subunit mRNA were found to have a similar distribution to the NOS cells. Double labeling for NOS immunoreactivity and NKA alpha-subunit mRNA revealed cellular colocalization of NKA alpha-subunit mRNA and nNOS protein in putative chloride cells at the base of the lamellae and interlamellar space. Along the lamellae, some NOS- or NKA-immunoreactive cells possessed a relatively lower expression of NKA alpha-subunit mRNA in smolts. A clear increase in NADPHd staining in the gill was demonstrated from parr to smolt. The regulatory role of NO on gill NKA activity was studied in vitro using sodium nitroprusside (SNP; 1 mmol l(-1)) and PAPA-NONOate (NOC-15; 0.5 mmol l(-1)) as NO donors. Both SNP and NOC-15 inhibited gill NKA activity by 30% when compared to controls. The study shows that NO systems are abundant in the gill of Atlantic salmon, that NO may be produced preferentially by a constitutive NOS isoform, and suggests that NO influence on gill functions is mediated via intracellular, possibly both auto/paracrine, inhibition of Na(+),K(+)-ATPase activity in chloride cells. Furthermore, the increase in NADPHd in the gill during smoltification suggests a regulatory role of NO in the attenuation of the smoltification-related increase in Na(+),K(+)-ATPase activity prior to entering seawater.