A cDNA clone (designated as GP2-7) encoding a novel 5-hydroxytryptamine (5-HT) receptor was isolated from a guinea pig hippocampal library. The receptor shares amino acid homology within the hydrophobic domains with other cloned 5-HT receptor subtypes (34-48%). The sequence of GP2-7 is homologous to that described for a novel receptor previously cloned from a rat brain cDNA library and provisionally designated as 5-HT7. mRNA for GP2-7 was detected in cortical and limbic brain regions. Transiently expressed GP2-7 showed high-affinity binding to [3H]5-HT (pKi = 9.0) with the following rank order of affinities: 5-carboxyamidotryptamine (5-CT) > 5-HT = 5-methoxytryptamine (5-MeOT) > methiothepin > 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) > spiperone >> sumatriptan. Adenylyl cyclase activity in CHO-K1 cells transiently transfected with GP2-7 was stimulated by several analogues of 5-HT with the following order of potency: 5-CT > 5-HT = 5-MeOT > dipropyl-5-CT > 8-OH-DPAT. Methiothepin and spiperone were potent antagonists. Preliminary analysis suggests that GP2-7 closely resembles a receptor in the guinea pig hippocampus that exhibits a high affinity toward 5-CT.