Although it is well established that activated LH/human (h) CG receptor stimulates adenylyl cyclase activity (via the heterotrimeric stimulatory guanine nucleotide-binding protein, Gs) and in some cells stimulates phospholipase C activity, there is no evidence for a direct physical interaction between the LH/CG receptor and Gs or any other G protein(s). We conducted studies using cholera toxin (CTX) and pertussis toxin (PTX) to determine which G alpha proteins were associated with the LH/CG receptor in ovarian follicular membranes. Since hormone-dependent, CTX-catalyzed ADP ribosylation (AR) constitutes evidence that a G alpha protein is specifically associated with a receptor, CTX-catalyzed AR of membrane proteins was examined both in the presence and absence of guanine nucleotides to determine which G proteins exhibit hCG-dependent labeling by [32P]NAD. Results demonstrated the time- and hCG-dependent AR of both a 45-kDa protein and a 48/50-kDa doublet as well as a 40-kDa protein that was also sensitive to AR by PTX in a time- and hCG-dependent manner. Using anti-G protein antisera to specifically immunoprecipitate photoaffinity-labeled G proteins, we were able to identify the 45- and 48/50 kDa proteins as the short and long forms of Gs alpha and the 40-kDa protein as Gi alpha. A monoclonal anti-hCG antibody immunoprecipitated the activated LH/CG receptor along with the long and short forms of Gs alpha and Gi. These results suggest that a portion of Gi along with the long and short forms of Gs alpha are associated physically with the LH/CG receptor in ovarian follicular membranes.