Sex hormone-binding globulin (SHBG) production in humans has been thought to be stimulated by estrogens and thyroid hormone and inhibited by androgens. However, recent data indicate that SHBG production in vitro is stimulated by both androgens and estrogens. This study was designed to determine what other hormonal factors regulate SHBG production. Since hyperinsulinemia and hyperprolactinemia both occur in disease states in which low serum SHBG levels are found, the effects of insulin and PRL were compared to and/or studied in combination with estradiol (E₂,) T₄, and testosterone (T) in a human hepatoma cell line (Hep G2). Hep G2 cells were grown to near confluence in medium including 10% fetal calf serum, and then 72-h experimental incubations were carried out which used only fetal calf serum-free medium. Compared to control incubations, both insulin (10⁻⁸ mol/L) and PRL (10⁻⁸ mol/L) decreased SHBG production from 65.0 ± 0.6 (±se) to 46.8 ± 1.1 and 46.8 ± 1.2 nmol/108 cells, respectively (P < 0.01). Insulin also inhibited both E₂ and T₄-stimulated SHBG production. T stimulated SHBG production to the same degree as E₂. Finally, both E₂ and insulin significantly increased cell number, an important consideration when expressing the effect of a hormone on SHBG production in cultured cells. We conclude that insulin and PRL inhibit SHBG production and confirm that T₄, T, and E₂ stimulate SHBG production in vitro. These findings suggest that insulin and PRL may be important factors in the regulation of SHBG production in vivo. (J Clin Endocrinol Metab67: 460, 1988)