The Na+,K+-ATPase (the sodium pump) plays a crucial role in ion transport in the fish gill. An immunocytochemical method has been optimized, using the mouse monoclonal antibody IgG alpha5, raised against the alpha subunit of the avian sodium pump, to localize Na+,K+-ATPase in fish gill cells. The method appears to be successful for the immunolocalization of Na+,K+-ATPase in both paraffin-embedded gill tissue sections and primary cultures of fish gill epithelial cells. The immunostaining has demonstrated that Na+,K+-ATPase-positive cells are mainly localized on the primary lamellae, in the interlamellar region, which is in agreement with the distribution of ion-transporting cells, also called chloride cells, as shown by electron microscopy. Na+,K+-ATPase-positive cells have been demonstrated for the first time in primary cultures of gill epithelial cells. Comparative labeling studies of primary cultures have shown that sites of Na+,K+-ATPase-positive cells correspond to sites of cells labeled with dimethylaminostyrylmethyl-pyridiniumiodine, a fluorescent mitochondrial probe for ion-transporting cells. The immunocytochemical detection method for Na+,K+-ATPase in cells is proposed as an easy and specific Na+-transport-related method to characterize and localize ion-transporting cells in primary cultures and in tissue sections of fish gill epithelium.