Insulin has pleiotropic effects on the regulation of cellular growth, differentiation, and metabolism. The biochemical events ultimately leading to cell proliferation after insulin treatment have been demonstrated in detail by numerous research groups. However, depending on cell types, it has been shown that insulin has various effects on cell proliferation. Therefore, we attempted to more critically evaluate the effect of insulin on cell proliferation in 3T3 L1 fibroblasts. In this study, we investigated insulin's effect on cell proliferation by using [3H]thymidine incorporation, flow cytometry, and cell counting. In 3T3 L1 fibroblasts studied in 0.5% serum, insulin induced a two-fold increase in [3H]thymidine incorporation over at 48 h, and the maximal rate of DNA synthesis was observed during 8-12 h incubation. The flow cytometric analysis also showed that insulin increased the cell population in the S phase. After insulin treatment for 48 h, cell numbers increased approximately 45% in comparison with 0.5% serum control. Cell division was found to occur only once in 60 h after staining 3T3 L1 fibroblasts with carboxyfluorescein diacetate succinimidyl ester (CFSE). Taken together, this data indicates that insulin stimulated the transit from the G0/G1 to S phase, progressed the cell cycle through the G2/M phase, and increased the cell number. However, under our experimental conditions, cells divided only once in 60 h in the presence of insulin.