Using a variety of techniques, several laboratories have recently demonstrated the feasibility of using radiation-induced DNA strand breaks and/or DNA-protein crosslinks (DPCs) to detect and/or quantify hypoxic cells in tumors and normal tissues. However, if strand breaks and/or DPCs are to be used to estimate the hypoxic fraction or the fractional hypoxic volume of tumors and normal tissues, their formation as a function of the oxygen concentration near the DNA must be relatively independent of the biological properties of these cells. In the present study, the shape of the oxygen dependence curves and the K(m) values for radiation-induced strand breaks and DPCs were measured by alkaline elution for proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma, line 66. The sigmoidal shape of the oxygen dependence curves, the K(m) for strand breaks (approximately 0.027 mM) and the K(m) for the formation of DPCs (approximately 0.020 mM) were identical for the P and Q cells of line 66. Consequently, the proliferative status of these tumor cells had no measurable influence on the oxygen-dependent formation of radiation-induced strand breaks and DPCs. In addition, the percentage of the DNA retained on the filters after approximately 24 ml of elution without proteinase K in the lysis solution, a parameter equal to the sum of the strand breaks and DPCs that has been shown to be proportional to the percentage of hypoxic cells in the sample, was not significantly different for fully oxygenated or fully hypoxic populations from five tumor cell lines that varied in species, site of origin, proliferative status and/or properties of the proteins which are intimately associated with their DNA. These data indicate that the formation of radiation-induced strand breaks and DPCs depends predominantly on the oxygen concentration in the microenvironment around the DNA, and only minimally on the biological properties of the cells.