Corticosteroid binding sites with the characteristics of steroid receptors were detected with the synthetic corticosteroid, [3H]triamcinolone acetonide (TA), in monolayers of the rainbow trout fibroblast cell line, RTG-2. The sites had low capacity as saturation was achieved at approximately 5 nM. Scatchard plots of the data suggested a single population of high-affinity binding sites. The number of receptors per cell was approximately 20,000; the dissociation constant, 1 nM. Changes in [3H]thymidine incorporation and cellular morphology were monitored as potential corticosteroid-sensitive metabolic responses. Only cortisol and 11-deoxycortisol among 14 naturally occurring steroids and TA, fluocinolone acetonide, dexamethasone, and prednisolone among 6 synthetic corticosteroids inhibited [3H]thymidine incorporation and altered the morphology in RTG-2 cells. Two observations suggested that the corticosteroid receptor mediated these responses. The synthetic steroid, RU 38486, which is an antiglucocorticoid in mammals, did not elicit these responses, had a high affinity for the receptor, and blocked the ability of cortisol and TA to change [3H]thymidine incorporation and cellular morphology. Second, the affinity of various natural steroids for the receptor correlated with their ability to elicit a cellular response. Cortisol, and to lesser extent 11-deoxycortisol, showed strong affinity for the receptor. Cortisone, aldosterone, and the sex steroids had no affinity and did not elicit cellular responses.