Cyclic AMP formation was found to increase in mouse neuroblastoma N18TG2 cells exposed to 5-hydroxytryptamine (5-HT). This response was concentration-dependent with an EC50 value of 0.22 microM. Tryptamine and other tryptamine-related compounds were also agonists in this assay with a rank order of potency of 5-methoxytryptamine > 5-HT > tryptamine > 2-methyl-5-HT > 5-carboxamidotryptamine >> alpha-methyl-5-HT. (D)-Lysergic acid diethylamide and 2-bromo-lysergic acid diethylamide were partial agonists in this system with maximal responses of 44 and 34%, respectively, compared to 5-HT. 5-HT-stimulated cyclic AMP formation was inhibited by clozapine, mianserin and methiothepin with pA2 values of 6.6, 6.5 and 6.4, respectively. Radioligand binding studies using [125I]iodolysergic acid diethylamide revealed a binding site present at a density of 10.4 fmol/mg of protein, with an affinity for the ligand of 1.18 nM. In competition studies this binding site displayed a pharmacology similar to that defined in studies of cyclic AMP formation. The pharmacological profile of this receptor, characterized by both radioligand binding and functional coupling to adenylyl cyclase, does not correspond to that of any of the currently classified subtypes of 5-HT receptor, but is similar to the 5-HT receptor cloned recently from rat striatum and referred to as the 5-HT6 receptor. The N18TG2 cell line represents a useful model system in which this novel 5-HT receptor may be characterized fully.