The effects of cytochalasin D on basal and adrenocorticotropin (ACTH)-stimulated steroid production as well as stress fiber organization by bovine adrenocortical (BAC) cells grown in monolayer culture have been investigated. Corticosteroid and pregnenolone release was determined by use of radioimmunoassay. The addition of ACTH (1 nM) produced a 30-fold increase in steroid release (principally corticosterone) in a 2 h period. Cytochalasin D (1-10 microM) had no effect on ACTH-stimulated release of corticosteroids. Basal steroid release was elevated by cytochalasin D with lower concentrations being more effective. In addition, hormonal stimulation of pregnenolone formation, the initial step in cholesterol metabolism leading to corticosteroid production, was also unaffected by cytochalasin D (1-50 microM) addition. Observation of stress fibers by fluorescence microscopy using the probe NDB - phalladin revealed that cytochalasin D (10 and 50 microM) caused an aggregation of actin-containing filaments into stellate foci within the cytoplasm. This was confirmed by electron microscopic examination of the cells. ACTH, however, had no observable effect on stress fiber organization or cell morphology. These results differ from the inhibitory effect of cytochalasin on steroid production observed using Y-1 adrenal tumor cells and rat adrenal cells in primary culture. BAC cells in contrast to Y-1 adrenal tumor cells differ in non-esterified cholesterol content and their morphological response to ACTH treatment. We suggest that these differences may influence the lack of inhibition of cytochalasin D on ACTH-stimulated steroid release.