A positive relationship between receptor concentration and tissue responsiveness is an often-assumed and rarely tested principle in endocrinology. In salmonids, seasonal changes in levels of plasma cortisol and gill corticosteroid receptors (CRs) during the spring indicate a potential role for this hormone in the parr–smolt transformation. It is not known whether these seasonal changes result in alterations in gill responsiveness to cortisol. The relationship between CR concentration and tissue responsiveness was, therefore, examined in the gills of juvenile rainbow trout (Oncorhynchus mykiss). Gill CR concentration (B_max) and affinity (K_d) were assessed using a radioligand binding assay with the synthetic glucocorticoid triamcinolone acetonide. Gill responsiveness to cortisol was quantified by measuring in vitro Na⁺/K⁺-ATPase activity. Gill CR concentration was manipulated by stress or hormonal treatments. Repeated handling stresses resulted in a significant reduction in CR numbers. The decrease in CR B_max corresponded to a reduction in gill responsiveness to cortisol. Triiodothyronine, but not growth hormone, treatment was found to increase CR B_max significantly. The increase in CR numbers was correlated with a marked increase in gill responsiveness to cortisol. A significant positive linear relationship exists between the in vitro gill Na⁺/K⁺-ATPase activity response to cortisol and CR B_max (r²=0.614, P<0.001). We have demonstrated that binding sites for cortisol in the gills of rainbow trout have high affinity, high specificity and saturable binding and that the number of binding sites is correlated with the tissue response to cortisol.