In this study, [125I]iodocyanopindolol ([125I]ICYP), in the presence of isoproterenol, was used to label 5-hydroxytryptamine1B (5-HT1B) receptors in homogenates of the cortex, substantia nigra and caudate-putamen of the rat. The determination of the appropriate concentrations of isoproterenol required to block optimally beta adrenoceptors whereas producing minimal occupancy of 5-HT1B receptors was achieved by generating isotherms for isoproterenol at multiple concentrations of [125I]ICYP. When different concentrations of isoproterenol were used with increasing concentrations of [125I]ICYP, a linear Scatchard transformation of the saturation curve was achieved, even with ligand concentrations about 6-fold greater than the KD for [125I]ICYP. Competition for [125I]ICYP (100 pM) labeled binding sites by 15 serotonin agonists or antagonists was adequately described by a single site model, and the affinity of these drugs for the site labeled by [125I]ICYP was similar to that determined previously when using indirect methods to label 5-HT1B receptors. Serotonin itself showed high affinity for this binding site as did two antagonists, metergoline and methiothepin. By contrast, drugs thought to be selective for the 5-HT1A receptor (e.g., 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone and spiperone) showed very weak affinity for the binding site labeled with [125I]ICYP. The effect of nucleotide regulation on [125I]ICYP binding at 5-HT1B receptors also was evaluated. It was determined that GTP had little effect on the binding of [125I]ICYP, reducing total binding by only 15% and shifting the displacement curve of 5-HT by a factor of less than two. The regulation of 5-HT1B receptors, labeled by [125I]ICYP, also was evaluated. Intraventricular injections of 5.7-dihydroxytryptamine increased significantly the number of 5-HT1B receptors in the caudate-putamen; this treatment had no effect on 5-HT1B binding sites either in the cortex or substantia nigra. The regulatable binding site for [125I]ICYP in the caudate-putamen had a pharmacological profile very similar to that of the 5-HT1B binding site in the cortex. [125I]ICYP appears to be a useful ligand to measure 5-HT1B receptors in the brain of the rat. The localized increase in 5-HT1B receptors in the caudate-putamen after destruction of central serotonergic neurons might indicate that the majority of 5-HT1B receptors in this area of brain are not located on serotonergic nerve terminals.