The synchronized HeLa cells were used to study the effect of protein kinase A(PKA) inhibitor on the progression of S phase. Synchronized cells in S phase were obtained by the method of TdR double block through ³H-TdR incorporation assay. The PKA inhibitor typeⅢ obviously increased the level of ³H-TdR incorporation of S phase in HeLa cells. In contrast with control, the activity of thymidine kinase (TK) in S phase increased, too. It indicated that PKA played an inhibitory role in S phase progression of HeLa cells. With the method of Western blotting, the PKA inhibitor typeⅢ enhanced the level of CyclinA and PCNA, inhibited the expression of p21, which is a negative regulator of cell cycle, but had no effect on the expression of CDK2. The results showed that PKA could negatively regulate the S phase progression by affecting the level of CyclinA, PCNA and influencing the expression of p21 protein. This may be one of the molecular mechanisms which is involved in the negative regulation of S phase progression by PKA in HeLa cells.