We tested the hypothesis that mouse ATC1 and ATC7 cells, the first adrenocortical cell lines to exhibit a complete zona fasciculata (ZF) cell phenotype, respond to dynamic ACTH stimulation in a similar manner as the adrenal gland in vivo. Exploiting our previous in vivo observations that gene transcription within the steroidogenic pathway is dynamically regulated in response to a pulse of ACTH, we exposed ATC1 and ATC7 cells to various patterns of ACTH, including pulsatile and constant, and measured the transcriptional activation of this pathway. We show that pulses of ACTH administered to ATC7 cells can reliably stimulate a pulsatile pattern of transcriptional activity that is comparable to that observed in adrenal ZF cells in vivo. Hourly pulses of ACTH stimulate dynamic increases in CREB phosphorylation (pCREB) and transcription of genes involved in critical steps of steroidogenesis including signal transduction (e.g., MRAP), cholesterol delivery (e.g., StAR), and steroid biosynthesis (e.g., CYP11A1), as well as those relating to transcriptional regulation of steroidogenic factors (e.g., SF-1 and Nur-77). In contrast, constant ACTH stimulation results in a prolonged and exaggerated pCREB and steroidogenic gene transcriptional response. We also show that when a large dose of ACTH (100 nM) is applied after these treatment regimens, a significant increase in steroidogenic transcriptional responsiveness is achieved only in cells that have been exposed to pulsatile, rather than constant, ACTH. Our data support our in vivo observations that pulsatile ACTH is important for the optimal transcriptional responsiveness of the adrenal. Importantly, our data suggest that ATC7 cells respond to dynamic ACTH stimulation.