Interferon (IFN) has been shown to enhance the cytotoxic effects of 5-fluorouracil (5FUra) in colorectal cancer, and clinical trials with this combination resulted in higher response rate with respect to 5FUra alone. IFN is generally administered s.c. three times a week. This prolonged exposure could determine a block of tumor cells in the G0-G1 phase of the cell cycle, thus rendering tumor cells insensitive to 5FUra, an S-phase specific agent. In order to verify the presence of this block, 21 operable colorectal cancer patients were treated with IFN-alpha 2b at the dose of 3 megaunits every other day in the week before operation, while another 22 represented the control group. Samples of tumor tissue were taken at endoscopy and operation. [3H]Thymidine labeling index and flow cytometry were used to assess the S-phase fraction. In IFN treated patients, we found a significant statistical difference between the mean percentage of S-phase fractions evaluated either by labeling index (P = 0.00001) or by flow cytometry (P < 0.001). On the contrary, this difference was not present in the control group: labeling index, P = 0.06; flow cytometry, P = 0.08. Furthermore a significant increase in the G0-G1 phase of the cell cycle was found after IFN administration (P < 0.001) but not in the control group. Our results suggest that IFN reduces the S-phase fraction in colorectal cancer tumors. This action should be considered in the design of the 5FUra/IFN combination because it could decrease 5FUra activity, leading to a loss or a decrease in the advantage of 5FUra modulation by IFN.