Confluent bovine adrenal cell primary cultures respond to stimulation by adrenocorticotropin (ACTH) to produce steroids (initially predominantly cortisol and corticosterone) at about one-tenth of the output of similarly stimulated rat adrenal cells. The early events of steroidogenesis, following ACTH stimulation, have been investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-6-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide (AMG), even though cytochrome P-450scc only increases after 12 h. Mitochondrial-free cholesterol levels increased during the same time period (16.5-25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. More prolonged exposure to ACTH prior to addition of AMG caused the elevation in mitochondrial cholesterol to more than double, possibly due to enhanced binding capacity. Early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial-free cholesterol. The maximum rate of cholesterol transport to mitochondria in AMG-blocked cells was consistent with the maximum rate of cellular steroidogenesis. Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial-free cholesterol. Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8) M), for 24 h prior to ACTH addition decreased the rates of ACTH- and cAMP-stimulated steroidogenesis but did not affect the basal rate. In both cases, the effectiveness of TCDD increased with time of exposure to the stimulant. Although cholesterol accumulated in the presence of ACTH and AMG (13-28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13-8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). Since even in the absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h) TCDD pretreatment may increase the presence of a protein(s) that cause this mitochondrial-cholesterol depletion following stimulation by ACTH or cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)