The physiological effects of ovine prolactin (oPRL) and recombinant rainbow trout prolactin (rbtPRL) on cultured gill epithelia derived from freshwater rainbow trout were assessed. Epithelia composed of either pavement cells only (single seeded inserts, SSI) or both pavement and mitochondria-rich cells (double seeded inserts, DSI) were cultured in media, supplemented with doses of oPRL ranging from 10 to 100 ng/ml. Under symmetrical culture conditions (L15 media apical/L15 media basolateral), oPRL had no effect on transepithelial resistance, paracellular permeability (assessed with PEG-4000), or Na(+) and Cl(-) transport across both preparations of cultured gill epithelia. Under asymmetrical conditions (freshwater apical/L15 media basolateral), SSI epithelia treated with oPRL (10 and 50 ng/ml), in comparison to comparably treated epithelia receiving no oPRL, exhibited a greater increase in the transepithelial resistance, particularly during the first 12h of freshwater exposure, no difference in paracellular permeability and Na(+)-K(+)-ATPase activity, and lowered net Na(+) flux rates (i.e., reduced basolateral to apical loss rates). These reflected reduced unidirectional efflux rates. The PRL effect appeared to be mainly a reduction in transcellular permeability. SSI epithelia treated with rbtPRL (10 ng/ml) exhibited similar patterns of response to those treated with oPRL. Na(+)-K(+)-ATPase activity increased in DSI epithelia treated with oPRL; however, oPRL did not stimulate ion uptake across either SSI or DSI epithelial preparations. The data demonstrated that, as the sole hormone supplement for cultured gill epithelia, PRL did not promote active ion uptake. However, the observed PRL-induced alterations in cultured gill epithelial physiology were consistent with the in vivo actions of PRL on the gills of freshwater teleost fish.