An affinity-chromatography column was used to isolate and purify 5-hydroxytryptamine (serotonin, 5-HT) receptors from bovine brain frontal cortex. The affinity ligand lysergic acid ethylamidoethylbromide was synthesized and coupled to an agarose matrix via a thioether bond. Receptors in the crude cortical membrane fragments were solubilized using 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), affinity purified, and reconstituted into lipid vesicles. [3H]5-HT binding analysis indicates a single class of high-affinity binding site (Kd, 16.9 nM) that was reconstituted. 5-Methoxytryptamine, a competitor for high-affinity serotonin sites, inhibited this binding and showed a Ki of 27.4 nM. Ketanserin, a high-affinity ligand for 5-HT2 type receptors, was ineffective in displacing [3H]5-HT binding at concentrations up to 4 microM indicating a 5-HT1 receptor as the primary receptor type isolated. The average specific activity of 359 pmol/mg in the reconstituted fractions is an enrichment of 1062-fold over crude membrane fragments. Sodium dodecyl-sulfate electrophoresis showed the presence of four proteins in the reconstituted vesicles with approximate relative Mr values of 63,000, 70,000, 81,000, and 94,000.