The peptide hormone ACTH stimulates lipolysis and suppresses leptin production in adipocytes via the G protein-coupled receptor, melanocortin 2 receptor (MC2-R). We have shown previously that peroxisome proliferator-activated receptor-gamma2 is the primary factor responsible for transactivation of the already identified murine MC2-R promoter in the differentiating 3T3-L1 adipocyte cell line. In this study we show that despite the activity of this promoter being transient during differentiation, MC2-R message remains elevated at later time points during adipogenesis. Analysis of the late transcripts reveals that they initiate from a transcriptional start site in the first intron of the murine MC2-R. The genomic sequence upstream of this start site acts as an adipocyte-specific promoter whose activation is delayed in differentiation, compared with the upstream promoter. A CCAAT/enhancer-binding protein binding site, 87 bp upstream of the transcriptional initiation site, is necessary for the activity of this promoter, and protein binding analyses reveal that this site is bound by CCAAT/enhancer-binding protein factors. Real-time PCR analysis of mRNA initiating from the two start sites shows that there is a switch in promoter usage from the 5' to the 3' promoter around d 5, indicating the complex regulation of the murine MC2-R during adipogenesis.