Adipocytes play an important role in normal physiology as a major site for systemic energy homeostasis. In disorders such as diabetes, adipocyte function is markedly altered. In this study, we investigated the effect of pioglitazone, a novel antidiabetic agent known to lower plasma glucose in animal models of diabetes mellitus, on cellular differentiation and expression of adipose-specific genes. Treatment of confluent 3T3-F442A preadipocyte cultures for 7 days with pioglitazone (Pio; 1 microM) and insulin (Ins; 0.17 microM) resulted in > 95% cell differentiation into lipid-accumulating adipocytes in comparison with 60-80% cell differentiation by treatment with either agent alone. Analysis of triglyceride accumulation showed increases of triglyceride content over time above untreated preadipocytes by treatment of the cells with Ins, Pio, and especially with Ins + Pio. Basal glucose transport, as measured by cellular uptake of 2-deoxy-D-[14C]glucose, was likewise enhanced in a time-dependent manner by treatment of preadipocytes with Ins, Pio, or Ins + Pio, such that a synergistic effect resulted from the combined treatment with both agents. It was further determined that RNA transcript abundance for genes encoding glucose transporters GLUT-1 and GLUT-4, as well as the adipose-specific genes encoding adipsin and aP2, were increased by the Ins, Pio, or Ins + Pio treatment. Taken together, these findings indicate that pioglitazone is a potent adipogenic agent. By promoting differentiation, this agent may move cells into a state active for glucose uptake, storage, and metabolism.