Three widely studied cell lines were used to examine the nature of the G1 arrest induced in human tumor cells by ionizing radiation and its relation to p53 status. Cell lines MCF-7 and RKO express wild-type p53, whereas HT29 expresses mutant p53. Exponentially growing cells were irradiated with 6 Gy, and the progression of G1 cells into S phase was monitored at regular intervals by flow microfluorimetric and continuous labeling autoradiographic techniques. In some experiments, cells were incubated with Colcemid prior to irradiation in order to block them in mitosis and to prevent the accumulation of cells in the second post-irradiation G1 phase. No evidence of a significant arrest at the first post-irradiation G1-S checkpoint was observed in any of the three cell lines. These results suggest that p53 function alone does not control the progression of irradiated human tumor cells from G1 into S during the first post-irradiation cell cycle. In particular, we found no evidence that radiation induced a prolonged G1 arrest in tumor cells expressing wild-type p53 as has been reported by some investigators.