The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of cortisol and prolactin as media supplements. Epithelia were of the double-seeded insert (DSI) type containing both pavement cells (PVCs) and mitochondria-rich cells (MRCs), and were grown in Leibovitz's L-15 media on filters in allowing exposure to different apical media conditions. Experiments were carried out in two series after 7-9 days symmetrical (L15 apical - L15 basolateral) culture. In both series, 100% L15 was maintained as the basolateral medium throughout and supplemented with physiologically relevant doses of either prolactin (50 ng ml^-1), cortisol (500 ng ml^-1), or cortisol + prolactin (500 ng ml^-1 + 50 ng ml^-1, respectively). In Series 1, epithelia were exposed to progressively diluted apical media (100%, 75%, 50%, 25%, 12.5% L15, and FW) at 24-h intervals. The preparations retained integrity (high transepithelial resistance, TER: low ion efflux rates) during this prolonged dilution protocol. Cortisol, or cortisol + prolactin, resulted in a greater TER and reduced ion efflux rates during dilution, likely an effect on junctional permeability of PVCs, but did not promote active Na⁺ and Cl^- uptake from apical FW. In Series 2, epithelia were directly exposed to apical FW and ion fluxes measured over the first 6 h. Under these conditions, cortisol or cortisol + prolactin promoted active uptake of both Na⁺ and Cl^- simultaneously from apical FW, probably attributable to actions on the MRCs. However, Na⁺, K⁺-ATPase activities were not significantly altered by any of the treatments in either series. Overall, prolactin alone did not appear to promote FW adaptation, but exhibited synergism with cortisol. These results provide further support for the cultured DSI epithelium as an in vitro model for ion transport in FW fish.