Binding of lutropin/choriogonadotropin to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. The mechanism underlying the generation of this bifurcating signal is presently not known. To analyze the coupling mechanism of the LH receptor, activated G proteins were labeled with [α-32P]GTP azidoanilide and identified by selective immunoprecipitation. In membranes of bovine corpora lutea and of L cells stably expressing the murine LH receptor (LHR cells), human chorionic gonadotropin (hCG) led to incorporation of the label into α(s) and α(i2). Stimulation of LHR cells or of L cells expressing the M5 muscarinic receptor (LM5 cells) with the respective agonist resulted in activation of phospholipase C in both cell lines. However, α(q) and α11 were only labeled upon stimulation of the M5 muscarinic receptor. Agonist-induced Ca2+ mobilization and inositol phosphate accumulation were partially sensitive to pertussis toxin, and the expression of the βγ-stimulable phospholipase C isoforms β2 and β3 could be demonstrated in LHR cells. Overexpression of phospholipase C-β2 led to increased hCG-stimulated inositol phosphate accumulation, and expression of a β-ARK1 C-terminal polypeptide effectively suppressed hCG-mediated phosphatidylinositol hydrolysis. Thus, the LH receptor couples to both G(s) and G(i), and βγ- subunits released from either G protein contribute to the stimulation of phospholipase C-β isoforms.