In this study, we investigated the effect of steroids on guinea pig and bovine adrenal steroidogenesis, especially 21-hydroxylase activity. Analysis of guinea pig adrenal steroids indicated the presence of high concentrations of androstenedione in the guinea pig adrenal; furthermore, in vitro studies using guinea pig adrenal cortex cells in primary culture confirmed that androstenedione is one of the major C19 steroids produced and secreted. The direct action of steroids on steroid production by adrenal cells was investigated. Our data indicate that steroids themselves increase C19 steroid synthesis and inhibit glucocorticoid production by guinea pig adrenal cells without affecting gene expression for steroidogenic enzymes. Incubation of a series of C19 steroids, namely, androstenedione, with guinea pig adrenal cell cultures demonstrated that the decrease in 21-hydroxylase activity is largely independent of the androgenic activity of C19 steroids. RU38486, a synthetic C18 steroid possessing a 4-ene-3-ketosteroid with an aryl group at position 11 and a very low affinity for the androgen receptor, also irreversibly altered 21-hydroxylase activity. An effect of RU38486 on 21-hydroxylase activity was also demonstrated in bovine adrenal cells. Further studies with bovine adrenal cells showed that the decrease in 21-hydroxylase activity induced by RU38486 was accompanied by a small but significant inhibition of P450c21 protein levels at both basal and ACTH-stimulated levels. In summary, our data indicate that alteration of 21-hydroxylase activity by steroids is likely due to a direct action on P540c21 protein, and the levels of androstenedione in the adrenal are high enough to inhibit 21-hydroxylase activity.