In this study, to discuss the importance of the cell cycle distribution in cell-based in vitro toxicity mechanism studies, diethyl sulfate (DES) was selected as a model chemical that induced the alteration of the cell cycle distribution in human bronchial epithelial cell line 16HBE 14o- (HBE) cells. Cells were treated with various concentrations of DES, cell proliferation and apoptosis were then determined. The results showed that DES concentration-dependently inhibited HBE cells proliferation and induced apoptosis. When cells were treated with 2.0 mM of DES for 20 or 28 h, significant S and G2/M phase accumulation was observed. Then, the relative cellular levels of Cdk4, p-Cdk2 (Thr160), Cyclins A and B1 in DES-treated HBE cells at 20 and 28 h were determined by two ways. The differences of the cell cycle distribution between DES and control groups were ignored in one way and eliminated by using flow cytometric cell sorting in the other. The results obtained by the two ways were quite different, which indicated that the cell cycle distribution might result in confounding if it was significantly different between the treated and control groups. Therefore, we propose that the cell cycle distribution should be given more consideration in cell-based in vitro toxicological studies.