Gonadotropin binding to the adult Leydig cell activates a GTP binding protein that interacts with adenylate cyclase to increase cAMP production within the cell. The increased production of cAMP stimulates steroidogenesis and leads to an increase in testosterone production and secretion. The fetal Leydig cell responds to LH with an increase in cAMP and testosterone production as early as 15.5 days of gestation, although the specific mechanism of transmembrane signaling has not been characterized. Fetal rat testis cells from 13.5-20.5 days of gestation were treated with dibutyryl cAMP (dbcAMP), cholera toxin, and hCG to determine the onset of steroidogenesis stimulation by activation of each moiety in the transmembrane signaling system of the fetal Leydig cell. Maximal stimulation at each age from 14.5 through 20.5 days of gestation was achieved with 1 mM dbcAMP, 500 ng/ml cholera toxin, or 10 ng/ml hCG. At 13.5 days of gestation, fetal testes did not produce any testosterone. These findings indicate that a cholera toxin-sensitive, stimulatory guanine-nucleotide regulatory protein is functional in the fetal Leydig cell as early as 14.5 days of gestation. The LH receptor becomes functional in the transmembrane signaling system of the fetal Leydig cell at 14.5 days of gestation.