The nuclear hormone receptor peroxisome proliferator activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that specifies formation of the adipocyte lineage. PPARgamma also serves as a primary target for the treatment of type 2 diabetes, illustrating both its medical relevance as well as the need to understand fundamental aspects of PPARgamma expression and function. Here, we characterize molecular changes that occur at the PPARgamma2 promoter within the first several hours of adipocyte differentiation in culture. Our results demonstrate that changes in chromatin accessibility at the PPARgamma2 promoter and occupancy of the promoter by the c-Fos transcription factor occur within an hour of the onset of differentiation, followed closely by the binding of the CCAAT/Enhancer Binding Protein beta (C/EBPbeta) transcription factor. All three events show a remarkable dependency on protein kinase A (PKA) activity. These results reflect novel requirements for the PKA signaling pathway and reinforce the importance of PKA function during the onset of adipocyte differentiation. J. Cell. Physiol. (c) 2010 Wiley-Liss, Inc.