Peroxisome proliferator-activated receptor (PPAR)-gamma plays an important role in adipogenesis. However, the functions of PPAR-gamma in differentiated adipocytes have remained unclear. The role of PPAR-gamma in mature 3T3-L1 adipocytes was therefore investigated by overexpression of a dominant negative mutant of this protein (PPAR-gamma-DeltaC) that lacks the 16 COOH-terminal amino acids and that has been shown to prevent the thiazolidinedione-induced differentiation of 3T3-L1 cells into adipocytes. Overexpression of PPAR-gamma-DeltaC in mature 3T3-L1 adipocytes by adenovirus gene transfer resulted in a decrease in both cell size and intracellular triglyceride content, an increase in the extent of lipolysis, and a reduction in the rate of free fatty acid uptake. Furthermore, overexpression of this mutant reduced the abundance of mRNAs for several key enzymes that contribute to triglyceride and free fatty acid metabolism as well as the amounts of GLUT4, insulin receptor, insulin receptor substrate (IRS), and C/EBPalpha mRNAs. It also reduced both the concentration of IRS2 and the insulin-stimulated glucose uptake. These results suggest that PPAR-gamma plays an important role in mature 3T3-L1 adipocytes at least in part by maintaining the expression of genes that confer the characteristics of mature adipocytes.