The potential role of satellite cells in mediating the effect of trenbolone [17 beta-hydroxyestra-4,9-11-trien-3-one (TBOH)] on skeletal muscle hypertrophy was examined. Young female Sprague-Dawley rats received TBOH injections daily for 2 weeks; growth, body composition, and the composition of selected muscles were assessed. Treated rats grew more rapidly and deposited less body lipid and more protein. The semimembranosus muscle from treated rats was larger and had approximately 60% more DNA per muscle than muscles from control rats. The addition of trenbolone directly to the medium of cultured satellite cells did not stimulate cell proliferation, nor did it augment the stimulatory response of these cells to fibroblast growth factor (FGF) or insulin-like growth factor I (IGF-I). In contrast, satellite cells cultured from TBOH-treated rats exhibited greater proliferative responses to FGF and IGF-I than satellite cells from control rats. In addition, serum from TBOH-treated rats stimulated greater cell proliferation in satellite cell cultures than serum from control rats. These experiments suggest that one possible mechanism responsible for the ability of TBOH to stimulate skeletal muscle hypertrophy may be through enhanced proliferation and differentiation of satellite cells as a result of the increased sensitivity of these cells to IGF-I and FGF.