Indirect evidence suggests that glucocorticoid hormones may act through cellular receptors to play a neuromodulatory role in the teleost CNS. We now report our findings on the use of [ 3 H]dexamethasone (DEX) to identify hypothalamic glucocorticoid receptors (GRs) in juvenile rainbow trout,Oncorhynchus mykiss.Hypothalamic cytosol was incubated with [ 3 H]DEX under various experimental paradigms with incubations terminated by addition of dextran-coated charcoal; following immediate centrifugation, a sample of bound [ 3 H]DEX (supernatant) was collected and assessed for 3 H content. [ 3 H]DEX binding was tissue dependent between 0.5 and 2.0 hypothalamus equivalents per tube (1.0 to 4.7 mg protein, respectively). Specific binding (B SP ) increased with time for 1.5 h and remained relatively constant for an additional 2.5 h; the calculated association rate constant was 2.23 × 10 8 M −1 × min −1 . EquilibriumB SP was dissociated by addition of a 5000 M excess cortisol with an accompaningt 1/2 of 1.25 h and dissociation rate constant of 0.553 min −1 .B SP was saturable with a calculated equilibriumK d andB MAX of 1.22 nM and 296 fmol/mg protein, respectively.B SP was displaced under equilibrium conditions by the corticosteroids, but to a lesser extent by the mineralocorticoid, estrogen, and progestin. The rank order of potency for [ 3 H]DEX displacement was DEX > cortisol ⪢ corticosterone > m triamcinolone = 11-deoxycortisol ⪢ aldosterone > progesterone >>> 17 β-estradiol. These properties of specifically bound [ 3 H]DEX indicate the presence of a GR, similar to the mammalian cytosolic GR, in the hypothalamus of juvenile rainbow trout.