Interstitial cells in the neonatal hamster do not respond to LH in vitro; however, side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17) enzyme proteins are expressed in these cells. The objective of the study was to evaluate whether the cAMP second messenger system was active in these cells and if cAMP upregulates the levels of CYP11A1 and CYP17 mRNA. Interstitial cells (ICs) were cultured for 96 h in the presence of 5% fetal bovine serum and then cultured in serum-free medium in the presence of LH, forskolin, or 8-Br-cAMP for 24 h. The accumulation of cAMP, progesterone, and androstenedione was measured by radioimmunoassay, whereas CYP11A1 and CYP17 mRNA levels were determined by a semiquantitative reverse transcription-polymerase chain reaction and Southern hybridization analysis. LH failed to induce either progesterone or androstenedione production; however, forskolin stimulated cAMP production by interstitial cells in a dose-dependent manner. Moreover, both forskolin and 8-Br-cAMP significantly elevated the levels of CYP11A1 and CYP17 mRNA and induced progesterone synthesis by the interstitial cell monolayer. Despite the increase in CYP17 mRNA levels by 8-Br-cAMP, no appreciable change was noted in androstenedione production. These results suggest that, in vitro, a fully functional adenylate cyclase system is present in cultured interstitial cells of the neonatal hamster and that cAMP can influence the expression of CYP11A1 and CYP17 genes; however, cultured cells do not appear to express LH receptors that are functionally linked to the adenylate cyclase system. Moreover, the translation of CYP17 mRNA may require additional factors, which may originate from maturing granulosa cells.