We studied the kinetics of ACTH induction of mitochondrial P450scc, P450c11 and their electron transport proteins, adrenodoxin, and adrenodoxin reductase in bovine adrenal cortex cells in primary culture. The mRNA levels of these enzymes increase and reach a peak within 3-12 h after ACTH addition. The protein levels of adrenodoxin reductase and P450scc show an increase only nearly 24 h after ACTH addition. After ACTH addition, the intracellular level of cAMP reaches maximal levels within 5 min, and then decreases gradually over 60 min. Hence, we examined the effect of a pulse of ACTH or cAMP analogs on enzyme and mRNA levels. Exposure of the cells to ACTH for 1-2 h was sufficient for maximal induction of the enzymes and P450scc mRNA. In contrast, the induction of the enzymes and the mRNA by cAMP analogs or forskolin required the continuous presence of these agents for over 12 h. But, these agents stimulated cortisol secretion to the medium quickly, indicating that they can activate some intracellular processes while not showing any effect on enzyme induction. The absence of any effect of prolonged cAMP pulses on enzyme and mRNA levels weakens the previous hypothesis that cAMP is the sole second messenger for the ACTH induction of steroidogenic enzymes in adrenal cortex cells. The inductive ability of a brief pulse of ACTH indicates that ACTH can rapidly initiate a series of reactions that result in enzyme induction many hours later.