The present study was carried out to clarify the direct effect of fatty acids (FAs) on chick (Gallus gallus) adipocyte differentiation in the absence of dexmethasone (DEX), a commonly used as strong inducer for adipocyte differentiation. Adipocyte differentiation was initiated by maintaining confluent cell in serum-free medium supplemented with FAs. Upon exposure to FAs, glycerol-3-phosphate dehydrogenase activity (GPDH) as adipocyte differentiation marker rapidly increased, and was significantly higher in chick adipocyte than in control cell. The morphology of the FAs-treated cell changed from fibroblast-like to polygon, and the cells accumulated many cytoplasmic lipid droplets as estimated by Oil red O staining. Neither insulin nor bovine serum albumin, as substitutes for serum, had an effect on chick adipocyte differentiation. The FAs-treated cell had a higher protein and mRNA expression levels for peroxisome proliferator-activated receptor-gamma (PPARgamma), a master regulator of differentiation, compared with untreated cell. In FAs-treated cell, the mRNA expression levels of adipocyte-specific genes, such as CCAAT/enhancer binding protein-alpha (C/EBP alpha) and adipocyte fatty acid-binding protein (aP2) were higher than in control cell. These results indicated that FAs, but not DEX, are essential inducers for chick adipocyte differentiation by elevating PPARgamma expression.