Gonadal steroids in the salmonid brain, acting through cellular receptors, may be responsible for the modulation of neuronal activity and organization of reproductive behaviors. We report our findings on the use of [3H]17beta-estradiol (E2) to identify intracellular estrogen receptors (ERs) in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss. Specific binding (B(SP)) of [3H]E2 was tissue dependent between 0.5 and 2.25 hypothalamus equivalents for cytosol and nuclear extract preparations, respectively. B(SP) in cytosol fractions increased with time and reached maximum levels (4.18 nM) at 2.5 h incubation; by contrast, B(SP) in nuclear extract increased with time to achieve maximum levels (3.9 nM) by 2 h incubation. The association rate constants (k(+1)) for cytosol and nuclear extract preparations were 1.10 +/- 0.02 x 10(8) M(-1) min(-1) and 1.27 +/- 0.04 x 10(8) M(-1) min(-1), respectively. Equilibrium bound B(SP) dissociated from cytosol preparations with a half life (t1-2) of 42 min and a dissociation rate constant (k(-1)) of 1.01 +/- 0.03 min(-1). B(SP) dissociated from nuclear extract preparations with a t1-2 = 45 min and k(-1)= 0.92 +/- 0.01 min(-1) x B(SP) was saturable in both extract preparations with a calculated equilibrium dissociation constant (Kd) of 1.46 +/- 0.1 nM (cytosol) and 2.37 +/- 0.2 nM (nuclear), and a maximum number of binding sites (B(MAX)) of 50.85 +/- 3.2 fmol mg(-1) protein and 61.74 +/- 2.65 fmol mg(-1) protein, respectively. In both preparations, B(SP) was differentially displaced by structurally similar compounds with a rank order of potency of E2 > estrone > estriol > 17alpha-ethynyl estradiol > testosterone >> progesterone = tamoxifen >> cortisol > dexamethasone >> > beta-sitosterol. These properties of specifically bound [3H]E2 suggest the presence of an ER in the hypothalamus of juvenile rainbow trout comparable with ERs identified in salmonid liver.