A method to culture tissue explants of the intestine from freshwater-adapted sockeye salmon (Oncorhynchus nerka) was developed to assess possible direct effects of cortisol on Na⁺, K⁺-ATPase activity. As judged by several criteria, explants from pyloric ceca and the posterior region of the intestine remained viable during short-term (6 days) culture, although Na⁺, K⁺-ATPase activity declined and basolateral components of the enterocytes were observed to be partially degraded. Addition of cortisol to the culture medium maintained Na⁺, K⁺-ATPase activity (over 2-12 days) above that of control explants and, in some cases, similar to levels before culture. The response to cortisol was dose-dependent (0.001 - 10 µg/ml). Within the physiological range, the response was specific for cortisol and showed the following hierarchy: dexamethasone cortisol > 11-deoxycortisol > cortisone. Insulin maintained Na⁺, K⁺-ATPase activity over controls in explants of ceca, but not posterior intestine. To compare in vivo and in vitro responses, slow-release implants of cortisol (50 µg/g) were administered to salmon for 7 days. This treatment elevated plasma cortisol levels and stimulated Na⁺, K⁺-ATPase activity in both intestinal regions. The results demonstrate that the teleost intestine is a direct target of cortisol, this corticosteroid protects in vitro functionality of Na⁺, K⁺-ATPase, and explants retain cortisol-responsiveness during short-term culture.