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The long-term effects of dibutyryl cyclic AMP [(Bu)2-cAMP] on steroidogenesis in bovine adrenocortical cells maintained in primary culture were investigated. During the first 36 h, total steroid production by cells incubated with (Bu)2-cAMP increased progressively. Thereafter, however, there was a marked fall in steroid output. During the first 36 h adrenocortical cells incubated in the presence of (Bu)2-cAMP produced substantially more C19-steroids and 17 alpha-hydroxylated C21-steroids than did cells incubated in the absence of (Bu)2-cAMP. By 48 h, however, such steroid secretion by cells incubated in the continued presence of (Bu)2-cAMP declined toward control levels. By contrast, the secretion of corticosterone and 11-deoxycorticosterone was consistently less by cells maintained in the presence of (Bu)2-cAMP than by cells maintained in its absence. These results suggest that refractoriness results, at least in part, from events which occur distal to the formation of cAMP. The action of ACTH and (Bu)2cAMP to promote the secretion of 17 alpha-hydroxylated C21-steroids and C19-steroids, on the other hand, appears to reflect an increase in the rate of cholesterol side-chain cleavage, as well as an increase in 17 alpha-hydroxylase and possibly also 17, 20-lyase activities.