The role of cytoplasmic microtubules in the process of steroidogenesis was examined in enzyme-dispersed and mechanically dispersed adrenocortical cells obtained from adult male rats. These cells were maintained in culture for periods of 2 h to 2 days and tested with various antimicrotubular drugs as well as ACTH and N6,O2-dibutyryl adenosine 3',5'-monophosphoric acid [(Bu)2cAMP]. The drugs tested, viz. colchicine, vinblastine, and nocodazole, were found to stimulate steroid synthesis in a dose-dependent fashion; the maximal degree of stimulation was found to be the same as that obtained with either ACTH or (Bu)2cAMP. When a maximal concentration of an antimicrotubular drug was mixed with a maximal concentration of either (Bu)2cAMP or ACTH, no additive effect on steroidogenesis was observed. The mixture of maximal concentrations of two antimicrotubular drugs produced a steroidogenic response that was less than when either drug was used alone. However, when submaximal concentrations of two antimicrotubular drugs were mixed, a synergistic response was noted. These antimicrotubular drugs are known to interfere with microtubular function by binding to specific sites on the tubulin protomer and inhibiting normal polymerization. Lumicolchicine, the photoinactivated form of colchicine, does not bind to tubulin and did not stimulate steroid synthesis. These studies support the notion that microtubules can influence the process of steroidogenesis.