5-Hydroxytryptamine1C (5-HT1C) recognition sites were characterized in rat spinal cord using [3H]mesulergine. In competition experiments with different 5-HT receptor agonists and antagonists, the rank order of drug potencies was consistent with drug affinities for the 5-HT1C receptor: mesulergine, mianserin, 5-HT greater than ketanserin, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane greater than 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)1H-indole, spiperone greater than 8-hydroxy-2-(di-n-propylamino)tetralin, pindolol. Bmax was 3.7 +/- 0.3 pmol/g and Kd 1.7 +/- 0.1 nM, with sites found in cervical, thoracic and lumbosacral cord. Inclusion of 20 nM spiperone to block potential 5-HT2 sites did not significantly alter drug affinities. There was a high correlation between drug affinities for [3H]mesulergine-labeled 5-HT1C sites in spinal cord and those reported in pig cortex (r = 0.94) and choroid plexus (r = 0.93), but poor correlation with 5-HT2 (high or low affinity states) or other 5-HT sites. Unlike [3H]mesulergine, the specific binding of [3H]5-HT, [3H]mianserin and [3H]ketanserin was low and no saturation studies could be performed with [3H]1-4-bromo-2-5-dimethoxy phenylisopropylamine. Limited competition studies suggest that [3H]5-HT labels 5-HT1C sites, [3H]ketanserin labels spinal 5-HT2 sites and [3H]mianserin labels both sites under the assay conditions studied, but the population of spinal 5-HT2 is small and not well characterized by these [3H]radioligands. In contrast, the population of spinal 5-HT1C receptors is substantial and [3H]mesulergine is the most useful [3H]radioligand for studies of spinal 5-HT1C sites.