in the first phase of this study, mammalian serotonin2-like (5-ht2-like) binding sites in juvenile rainbow trout (oncorhynchus mykiss) hypothalamus, were examined by radioligand binding assay using the tritiated analog of a selective serotonin antagonist, ketanserin ([3h]ketanserin), as the radioligand. specific [3h]ketanserin binding (bsp) to juvenile hypothalamus membrane was tissue-dependent where bsp increased linearly with tissue concentration. therefore, 1 hypothalamus-equivalent per tube (1100 ± 115 cpm/mg protein) was subsequently used throughout the rest of the first phase. in association experiments (n=5), bsp increased progressively with time to achieve equilibrium binding levels (1192+ 120 cpm/mg protein) which remained stable for at least 60 min thereafter; kobs, and k+i were 0.032 and 0.048 min-1 nm-1 respectively. this consistent, and relatively stable association of radioligand to the binding site indicates good stability of [3h]ketanserin binding to this binding site. in dissociation experiments, bsp completely dissociated within 20 min following addition of excess ketanserin; k_i, and t1/2 were 0.0803 min-1 and 8.7min, respectively. this pattern of [3h]ketanserin binding to this binding site is consistent with the association and dissociation kinetics of radioligand binding to a receptor. bsp was saturable (2500 ± 256 cpm/mg protein); scatchardcalculated values for the equilibrium dissociation constant (kd) and capacity (bmax) were 0.48nm, and 125 fmol/mg protein, respectively, indicating the presence of a finite population of high-affmity 5-ht2-like binding sites. bsp was differentially displaced by various competitors, with a rank order of potency of ketanserin = mianserin > ritanserin > 5-ht = spiperone » methiothepin mesylate > metergoline = doi > 2-methyl-5-ht > amethyl- 5-ht »»5-hiaa = reserpine. this rank order suggests that specific 5-ht2 agonists and antagonists displace specifically bound [3h]ketanserin more effectively compared to non-specific competitors, and in a manner comparable with the 5-ht2-like binding site of mammals. collectively, these findings provide pharmacological evidence for the existence of a 5ht2-like receptor subtype in the trout hypothalamus.