In the present study we examined the trout gill cell line, RTgill-W1, as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1-2 days (20-80 Ωxcm2) which was then maintained for more than 6days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6hours. Treatment with ethylenediaminetetraacetic acid (EDTA) induced a decrease in TER to <15% of pre-treatment level. Cortisol elevated TER after 12-72 hours in a concentration dependent manner and this rise was antagonized by growth hormone (Gh). The effects of three osmoregulatory hormones; Gh, prolactin and cortisol on mRNA expression of three tight junction proteins were examined: claudin-10e (Cldn-10e), Cldn-30 and zonula occludens-1 (Zo-1). Expression of cldn-10e was stimulated by all three hormones but with the strongest effect of Gh (50-fold). cldn-30 expression was stimulated especially by cortisol (20-fold) but also by Gh (4-fold). Finally, zo-1 was unresponsive to hormone treatment. Western blot analysis detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive to hormone treatments and may provide a useful model for in vitro study of some in vivo gill phenomena.