OBJECTIVE To apply the alkaline comet assay to the detection of radiobiologically hypoxic cells in solid tumors and normal tissues of mice, and to examine the influence of strand break repair on the oxygen enhancement ratio measured using the alkaline comet assay. METHODS In previous studies, we found that hypoxic fraction in squamous cell carcinomas growing in C3H mice could be reliably and easily measured using the alkaline comet assay. The comet assay applies fluorescence microscopy and image analysis to examine patterns of migration of deoxyribonucleic acid from individual cells embedded in agarose and exposed to an electric field. This method has sufficient resolution to detect subpopulations of hypoxic cells which show about 3 x fewer strand breaks than aerobic cells after irradiation. RESULTS Fast rejoining kinetics in vitro are comparable to those measured in vivo, and rejoining of strand breaks in hypoxic tumor cells occurs at a similar rate as rejoining in aerobic cells. Little residual damage was detectable using the comet assay in tumors 4-24 h following 15 Gy, allowing repeat measurements to be performed. Bone marrow and testis, but not liver, spleen, or jejunum contained a small fraction of hypoxic cells when mice breathed 10% oxygen during irradiation. CONCLUSIONS The comet assay confirms that some normal tissues may border on hypoxia. Rejoining of strand breaks occurs rapidly in both oxic and hypoxic cells so that the oxygen enhancement ratio remains relatively constant with time after irradiation. Interestingly, a smaller oxygen enhancement ratio was observed in tumors than was expected, probably as a result of the presence of acutely hypoxic cells.