The serotonin 5-HT(6) receptor, a G-protein-coupled receptor, displays high affinity for antipsychotic, antidepressant, and psychotropic drugs. We created a constitutively active form of the human 5-HT(6) receptor in order to probe the molecular domains of receptor activation and to determine if inverse agonist activities of antipsychotic drugs contribute to their clinical profile. Previous studies from our laboratory support a critical role for the c-terminal region of the third intracellular loop (il3) in the activation of G(q)-coupled serotonin receptors. In the present study, PCR-based mutagenesis was used to mutate serine 267 (S6.34) in the c-terminal region of il3 to lysine (S267K). The native and S267K 5-HT(6) receptors were expressed in COS-7 cells to study the functional effects of the mutation. The S267K receptor shows 10-fold higher affinity for serotonin than the native receptor and demonstrates agonist-independent activity. Clozapine decreased the basal activity of the S267K receptor to vector control levels. Therefore, we can conclude that the S267K mutation renders the 5-HT(6) receptor constitutively active and that clozapine is an inverse agonist at the mutant 5-HT(6) receptor. These results indicate that the c-terminal region of il3 of the G(s)-coupled 5-HT(6) receptor is a key domain for G-protein coupling, similar to the G(q)-coupled 5-HT receptors. The inverse agonist action of clozapine indicates that drugs displaying competitive antagonist activity at native 5-HT(6) receptors may display inverse agonist activity at the constitutively activated form of the receptor.