The purpose of the present study was to determine the effects of cortisol on the development of the freshwater chloride cell (CC), using flow cytometry. Scanning electron microscopy was used to determine the corresponding modifications in CC apical structure. Simultaneously, biochemical analyses were conducted to determine the activities of transport ATPases, mitochondrial enzymes (succinate dehydrogenase (SDH) and Mg(2+)-ATPase) and lactate dehydrogenase (Ldh). The effects of daily i.m. injection of 2 microg/g cortisol were compared with sham-injected freshwater-, control freshwater- and seawater-adapted fish. The hormone did not affect the activities of Ca(2+)-ATPases in CCs. However, it stimulated the proliferation and differentiation of the two freshwater CC subtypes (F1, 66+/-2.18% (s.e.m. ) and F2, 34+/-2.18%), in which the relative proportion of F1 CCs was transiently reduced in the first 5 days of treatment (F1, 53+/-1.83%; F2, 47+/-1.83%) but was then restored to a higher relative percentage on day 10 (F1, 70+/-1.42%; F2, 30+/-1.42%). Biochemically, it induced the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, SDH and Ldh, suggesting an increase in ion pumping and its associated metabolic activities. CCs from cortisoltreated fish demonstrated recessed apical morphology, accompanied by an increase in cell density (2012 to 2413/mm(2)). Nevertheless, the extent of cell proliferation and differentiation and the biochemical changes were significantly lower than those of seawater fish. Our results indicate that cortisol alone cannot stimulate a complete differentiation of freshwater CCs to seawater CCs. However, the respective roles of the two CC subtypes in freshwater and seawater environments are indicated.