This protocol describes how to reconstruct and culture the freshwater rainbow trout gill epithelium on flat permeable membrane supports within cell culture inserts. The protocol describes gill cell isolation, cultured gill epithelium formation, maintenance, monitoring and preparation for use in experimental procedures. To produce a heterogeneous gill epithelium, as seen in vivo, seeding of isolated gill cells twice over a two day period is required. As a consequence this is termed the double seeded insert (DSI) technique. Approximately 5-12 days after cell isolation and seeding, preparations develop electrically tight gill epithelia that can withstand fresh water on the apical cell surface. The system can be used to study freshwater gill physiology, as well as a humane alternative for toxicity testing, bioaccumulation studies and environmental water quality monitoring.