Several methods of establishing low O(2) conditions have been used in studies on the response of cultured cells to radiation and other agents. These methods, eg, gassing culture vessels with O(2)-free nitrogen with or without carbon dioxide or placing high cell-density suspensions in sealed glass ampoules to consume O(2) in the ampules, can be technically demanding and have experimental limitations. We introduce a simple, versatile, and reliable method of producing low O(2) conditions without special equipment or changes in culture conditions unrelated to hypoxia. The method is based on the ability of Oxyrase (Oxyrase, Inc., Mansfield, OH), membrane fragments prepared from Enterococcus coli, to consume O(2) in solution and is confirmed in the present study by 2 analytical methods. The effects of low O(2) conditions induced by Oxyrase on cellular responses to radiation and treatment with the bioreductive agent tirapazamine (TPZ) were examined with Chinese hamster V79 and human glioma U373 cells. Measured by clonogenic and MTT assays, these cells were less sensitive to radiation but more sensitive to TPZ in treatment media containing native Oxyrase than in media containing heat-inactivated Oxyrase. In addition, Oxyrase treatment increased the basal activity of mitogen-activated protein kinase (ERK1/2) but suppressed its activation induced by radiation. The results suggest that this method might also be useful for other in vitro cancer biologic investigations requiring a low O(2) condition.