G1 phase cell cycle arrest after exposure to ionizing radiation has been documented in cells with wild-type p53. The temporal location of this arrest within G1 phase, however, has not been determined. We have now used flow cytometric analysis of bromodeoxyuridine (BrdUrd)-labeled cells to obtain further information about the location of the G1 phase radiation checkpoint. Human fibroblasts were irradiated with gamma-rays and treated with colcemid to stop unlabeled G2 cells from entering the G1 phase. Analysis of BrdUrd incorporation revealed that 73% of G1 phase human lung fibroblasts remain in G1 phase after exposure to gamma-rays, thereby placing the G1 radiation checkpoint near the end of G1 phase. The location of the radiation checkpoint correlates with the reported increased expression of cyclin E, increased cyclin E/cdk2 kinase activity, and hyperphosphorylation of pRb in proliferating human fibroblasts.