The effects of PPAR-γ agonists, thiazolidinediones (TZDs), on preadipocytes isolated from rat mesenteric adipose tissue and murine cell line 3T3-L1 were compared using an in vitro cell culture system. After each cell formed a confluent monolayer under appropriate medial conditions, pioglitazone or troglitazone was applied at 10 μM to each medium for cell maturation. We observed morphological changes in each cell, especially the accumulation of lipid droplets in the cytoplasm, during the culture periods. At the end of culture, DNA content, triglyceride (TG) content and glycerol-3-phosphate dehydrogenase (GPDH) activity were determined. Adiponectin concentrations in each culture medium were also measured during appropriate experimental periods. Application of TZDs increased the DNA content, TG accumulation and GPDH activity in the 3T3-L1 cells but not in the mesenteric adipocytes. Although TG accumulation was unchanged, the number of lipid particles was decreased and the size of lipid particles in the mesenteric adipocytes was increased by TZD application. Although the TZDs increased adiponectin release from the 3T3-L1 cells, adiponectin release from mesenteric adipocytes was suppressed (P < 0.05). Thus, the effects of TZDs differed between the primary culture of mesenteric adipose cells and the line cell culture of 3T3-L1 cells. The source of adipocytes is an important factor in determining the action of TZDs in vitro, and particular attention should be paid when evaluating the effect of PPAR-γ agonists on adipose tissues.