Site-directed mutagenesis was performed to create a mutant serotonin 5-HT2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS-7 cells, the binding affinity of 5-HT for [3H]mesulergine-labeled 5-HT2C receptors increased from 203 nM (native) to 76 nM for S312F and 6.6 nM for S312K mutant receptors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 nM (native) to 28 nM for S312F and 2.7 nM for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [3H]5-HT and [3H]mesulergine labeled the same number of S312K mutant receptors and 5'-guanylylimidodiphosphate had no effect on [3H]5-HT binding. These results indicate that serine --> lysine mutation at amino acid no. 312 produces an agonist high-affinity state of the 5-HT2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.