The purpose of these studies was to determine whether the antiobesity actions of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) observed in vivo are due to an influence on proliferation and/or differentiation in monolayer cultures of 3T3-L1 preadipocytes. For the proliferation study (Exp. 1), cells were grown in plating medium containing DHEA at 0, 5, 25, 50, or 100 microM for 1-4 d. DHEAS was added at the 100 microM level only. For the differentiation study (Exp. 2), cultures were grown in plating medium containing DHEA at 0, 5, 30, 60, 120, or 240 microM for 2-6 d. DHEAS was added at the 240 microM level only. In Exp. 3, the effect of DHEA on mature adipocytes was determined by exposing adipocytes grown in plating medium to DHEA at 0, 75, 125, and 250 microM for 1-4 d. In Exp. 1, preadipocyte proliferation decreased as the level of DHEA increased in cultures of 3T3-L1 cells. DHEAS had no effect on preadipocyte proliferation. The antiproliferative effect of DHEA was partially reversed by the addition of 1 microM mevalonic acid to proliferating cultures containing 25 microM DHEA. In Exp. 2, preadipocyte differentiation decreased as the level of DHEA in the cultures increased. In contrast, neither DHEAS nor mevalonic acid treatment influenced preadipocyte differentiation decreased as the level and duration of DHEA treatment increased in cultures of mature adipocytes. These data support the hypothesis that DHEA, but not DHEAS, is the active form of the steroid that attenuates obesity via altering preadipocyte proliferation and differentiation. The addition of 1 microM mevalonic acid to cultures of 3T3-L1 preadipocytes partially reversed DHEA's antiproliferative effects.