Studies of the regulation of 5-hydroxytryptamine (5-HT2) receptors in vivo have generated anomalous and sometimes contradictory results. In particular, administration of antagonists unexpectedly results in a reduction in the density of 5-HT2 receptors. P11 cells, which express a high density of 5-HT2 receptors coupled to phosphoinositide hydrolysis, were used to investigate the regulation of receptors in vitro by agonists, partial agonists, and antagonists. (+/-)-2,5-Dimethoxy-4-iodophenylisopropylamine (DOI) and (+)-lysergic acid diethylamide (LSD) caused marked reductions in the density of 5-HT2 receptors as has been observed in vivo. Down-regulation was prevented by coincubation with ketanserin. The decrease in the density of 5-HT2 receptors after exposure to 5-HT, LSD, or DOI was time dependent and was not a consequence of residual drug in binding assays or irreversibly bound drug. The ability of 5-HT, DOI, and LSD to down-regulate 5-HT2 receptors was not proportional to the ability of these compounds to stimulate phosphoinositide hydrolysis. Ketanserin and mianserin, antagonists which cause paradoxical decreases in the density of 5-HT2 receptors in vivo, did not alter the density of 5-HT2 receptors on P11 cells, even after prolonged incubation with drug. Results of the current studies, which demonstrate agonist- but not antagonist-induced down-regulation of 5-HT2 receptors, lead to the conclusion that the ability of ketanserin and mianserin to down-regulate receptors in vivo is the result of indirect actions of these drugs and is unlikely to be a direct consequence of receptor occupancy by antagonists.