This report summarizes the genome-wide effects of ACTH on transcript accumulation in mouse adrenal Y1 cells and the relative contributions of the cAMP-, protein kinase C- and Ca(2+)-dependent signaling pathways to these actions of the hormone. ACTH affected the accumulation of 1386 transcripts, a much larger number than previously appreciated. The cAMP signaling pathway accounted for approximately 56% of the ACTH effects whereas the protein kinase C- and Ca(2+)-dependent pathways made smaller contributions to ACTH action. Approximately 38% of the ACTH-affected transcripts could not be assigned to these signaling pathways and thus represent candidates for regulation via other mechanisms. The set of ACTH-regulated transcripts included clusters with functions in steroid metabolism, cell proliferation and alternative splicing. Collectively, our results suggest that Y1 adrenal cells undergo extensive remodeling upon prolonged stimulation with ACTH. The functional implications of ACTH on alternative splicing are explored.