The adipocyte differentiation process involves a cascade of transcriptional events that culminates in the expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) and CCAAT/enhancer binding protein-alpha (C/EBPalpha). These adipogenic transcription factors regulate the expression of genes necessary for the development of mature adipocytes in mammals. The current study was undertaken to identify regulatory factors that affect adipogenesis and to analyze species-specific mRNA expression of factors involved in chicken adipocyte differentiation. We developed a system for differentiation of chicken (Gallus gallus) adipocytes in culture using medium containing 500 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 20 microg/mL bovine insulin, 300 microM oleate, and 10% fetal bovine serum. The rapid differentiation of cells to mature adipocytes in this culture system was verified by observed increases in adipocyte fatty acid-binding protein (aP2) expression, glycerol-3-phosphate dehydrogenase (GPDH) activity and intracellular triglyceride accumulation. In contrast, cells cultured in a differentiation medium without fatty acids did not differentiate into mature adipocytes. The expression profiles of genes involved in the regulation of adipocyte differentiation, such as PPARgamma, C/EBPalpha, beta, delta, sterol regulatory element binding protein-1 (SREBP-1), fatty acid synthase (FAS), lipoprotein lipase (LPL), and glucose transporters 1 and 8 (GLUT1 and GLUT8) were studied. Rapid increases in PPARgamma and aP2 expression were observed after 9 and 12 h of culture in differentiation medium, respectively. In contrast, the expression patterns of the other adipogenic genes only differed slightly from those previously determined for mammalian adipocytes. These results suggest that exogenous fatty acid is essential for adipocyte differentiation in chickens, and that PPARgamma is possibly a key regulator in the early stages of chicken preadipocyte differentiation.