(-)[125I]Iodocyanopindolol ([125I]CYP) labels rat brain membrane sites which display high affinity for several serotonergic and beta-adrenergic compounds. The binding of [125I]CYP to these serotonergic recognition sites was evaluated in the presence of 30 microM (-)isoprenaline in order to suppress binding to beta-adrenoceptors. [125I]CYP binds in rat cortex membranes rapidly, reversibly and stereoselectively to a finite number of recognition sites: Bmax = 180 fmol/mg, KD = 230 pM. Similar affinity values of [125I]CYP were obtained in membranes from rat hippocampus and striatum. Kinetic, saturation and competition experiments suggest that under these conditions [125I]CYP binds to a single serotonergic recognition site named 5-HT1B. The pharmacological profile of 5-HT1B sites is characteristic of a 5-HT1 binding site and shows the following rank order of affinity for agonists: RU 24969, (5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]1H-indole) greater than 5-CT, (5-carboxamidotryptamine) greater than 5-HT, (5-hydroxytryptamine, serotonin) greater than 5-OCH3-T, (5-methoxytryptamine) much greater than 2-CH3-5-HT, (2-methylserotonin) greater than 8-OH-DPAT, (8-hydroxy-2-(di-n-pro-pylamino)-tetralin). The rank order of affinity for antagonists is: (+/-)ICYP, ((+/- )-3-I-cyano-pindolol) greater than (-)21-009, (4-[3-ter-butyl-amino-2-hydroxy-propoxy]-indol-2-carbonic acid isopropyl ester) greater than (+)21-009 greater than (-)propranolol greater than metitepin greater than (-)pindolol much greater than ketanserin greater than spiroperidol greater than mesulergine. 5-HT1B recognition sites display low affinity for selective beta 1- and beta 2-adrenoceptor antagonists, e.g. atenolol, betaxolol, ICI 89-406 and ICI 118-551. The low affinity of 5-HT1B recognition sites for some 5-HT1A, 5-HT1C and 5-HT2 selective compounds (e.g. 8-OH-DPAT, mesulergine, ketanserin) suggests that 5-HT1B recognition sites are pharmacologically different from 5-HT1A, 5-HT1C and 5-HT2 recognition sites.