LH and insulin are postulated to jointly stimulate theca-cell androgen biosynthesis in patients with hyperthecosis or polycystic ovarian syndrome. To explore the mechanisms of putative LH and insulin steroidogenic synergy in primary culture of normal theca cells, we have implemented an in vitro serum-free monolayer culture system of Percoll-purified, porcine theca cells harvested from immature ovaries. Initial dose and time course analyses revealed that a maximally effective concentration of LH (100 ng/ml) or insulin (100 ng/ml) individually will drive androstenedione production (at 6 to 48 h) by 1.5- to 2.6- and 1.1- to 1.7-fold, respectively, while combined agonists act synergistically over the interval 12 to 48 h yielding a 3- to 4-fold joint effect. Coadministration of LH and insulin can augment theca-cell concentrations of CYP17 and StAR messenger RNA (mRNA) resulting in 3.4- to 3.9- and 3.8- to 4.1-fold increases at 24 to 48 h, respectively (P < 0.01). Combined LH and insulin stimulation also amplified the nuclear content of intron-specific heterogeneous nuclear (hn)RNAs encoding CYP17 and StAR. Insulin significantly enhanced LH-driven but not basal cAMP accumulation (14-18 vs. 3-5.5 pmol/microg DNA/12-48 h) (P < 0.01). A stable exogenous analog of cAMP, 8 Br-cAMP, mimicked LH's effect on steroidogenesis and StAR and CYP17 gene expression and with insulin stimulated StAR mRNA and hnRNA accumulation synergistically. However, unlike LH, 8 Br-cAMP did not synergize with insulin on theca-cell androstenedione biosynthesis or CYP17 mRNA and hnRNA expression. In summary, the present in vitro data identify molecular interactions of LH and insulin on StAR and CYP17 gene expression, thus establishing potent signaling interfaces between these distinct hormonal agonists in regulating theca-cell steroidogenesis.