ACTH regulates the steroidogenic capacity, size and structural integrity of the adrenal cortex through a series of actions involving changes in gene expression; however, only a limited number of ACTH-regulated genes have been identified and these only partly account for the global effects of ACTH on the adrenal cortex. In this study, a NIA 15K mouse cDNA microarray was used to identify genome-wide changes in gene expression following treatment of Y1 mouse adrenocortical cells with ACTH. ACTH affected the levels of 1275 annotated transcripts, of which 46% were up-regulated. The up-regulated transcripts were enriched for functions associated with steroid biosynthesis and metabolism; the down-regulated transcripts were enriched for functions associated with cell proliferation, nuclear transport and RNA processing, including alternative splicing. A total of 133 different transcripts, i.e. only 10% of the ACTH-affected transcripts, were represented in the categories above; most of these had not been described as ACTH-regulated previously. The contributions of protein kinase A and protein kinase C to these genome-wide effects of ACTH were evaluated in microarray experiments following treatment of Y1 cells and derivative protein kinase A-defective mutants with pharmacological probes of each pathway. Protein kinase A-dependent signaling accounted for 56% of the ACTH effect; protein kinase C-dependent signaling accounted for an additional 6%. These results indicate that ACTH affects the expression profile of Y1 adrenal cells principally through cAMP- and protein kinase A-dependent signaling. The large number of transcripts affected by ACTH anticipates a broader range of actions than previously appreciated.