We have reported that HT29 colon cancer cells, which are radiosensitized by fluorodeoxyuridine (FdUrd), exhibit a greater increase in cyclin E-dependent kinase activity and progress further into S phase in the presence of FdUrd than do SW620 colon cancer cells, which are only minimally sensitized by this drug (Cancer Res 56: 3203, 1996). Although these findings suggested that the ability to progress into S phase in the presence of FdUrd permits cells to be radiosensitized, we wished to test this hypothesis by attempting to drive SW620 human colon cells into S phase by transducing them with the HPV16-E7 gene. Two-parameter flow cytometry showed that E7-transduced cells progressed through S phase after radiation and FdUrd treatment more rapidly than SW620 parental cells. We found that E7-transduced SW620 cells were significantly radiosensitized by FdUrd (100 nmol/L, 14 hours) with an enhancement ratio for 2 clones of 1.47 +/- 0.03 and 1.51 +/- 0.14, compared with 1.24 +/- 0.04 in SW620 parental cells. These data strongly support the hypothesis that dysregulation of S-phase progression is an important factor in FdUrd-mediated radiosensitization.