Cell cycle checkpoints are among the multiple mechanisms that eukaryotic cells possess to maintain genomic integrity and minimize tumorigenesis. Ionizing irradiation (IR) induces measurable arrests in the G₁, S, and G₂ phases of the mammalian cell cycle, and the ATM (ataxia telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all three of these cell cycle phases. However, cells lacking ATM function exhibit both a defective G₂ checkpoint and a prolonged G₂ arrest after IR, suggesting the existence of different types of G₂ arrest. Two molecularly distinct G₂/M checkpoints were identified, and the critical importance of the choice of G₂/M checkpoint assay was demonstrated. The first of these G₂/M checkpoints occurs early after IR, is very transient, is ATM dependent and dose independent (between 1 and 10 Gy), and represents the failure of cells which had been in G₂ at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G₂ cells must be used to assess this arrest. In contrast, G₂/M accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after IR, is ATM independent, is dose dependent, and represents the accumulation of cells that had been in earlier phases of the cell cycle at the time of exposure to radiation. G₂/M accumulation after IR is not affected by the early G₂/M checkpoint and is enhanced in cells lacking the IR-induced S-phase checkpoint, such as those lacking Nbs1 or Brca1 function, because of a prolonged G₂ arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G₂ checkpoints appear to affect survival following irradiation. Thus, two different G₂ arrest mechanisms are present in mammalian cells, and the type of cell cycle checkpoint assay to be used in experimental investigation must be thoughtfully selected.