In the present study, we investigated the role of p16^INK4a in the inhibition of DNA synthesis stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) using RNA interference in primary cultured rat hepatocytes. The transfection of small interfering RNAs targeting p16^INK4a reduced the corresponding mRNA and protein expression by more than approximately 90% and 50%, respectively, at 24 h after transfection. In the cells transfected with p16^INK4a small interfering RNA, control, HGF, and EGF-stimulated DNA synthesis as assessed by ³H-thymidine incorporation increased by approximately 1.5-fold, 1.6-fold, and 1.7-fold, respectively, compared with that in the control small interfering RNA-transfected cells. These findings indicate that p16^INK4a plays a significant role in the inhibition of DNA synthesis.