Ischemia and hypoxia are major causes of renal failure and altered oxygen supply may affect renal responses to toxic chemicals. In vitro experiments were designed to evaluate the susceptibility of isolated proximal tubular (PT) and distal tubular (DT) cells from rat kidney to brief periods of oxygen deprivation and to assess how variations in oxygen supply affect chemical-induced cytotoxicity. Isolated cells were incubated for 1 hr in either oxygen (95% O2/5% CO2), air (21% O2), or nitrogen (95% N2/5% CO2) atmosphere. PT cells exhibited no injury due to brief oxygen deprivation whereas DT cells exhibited moderate, but significant injury, indicating that DT cells are more susceptible than PT cells to hypoxic injury. The cytotoxicity of chemicals that alter cellular redox status [i.e. tert-butyl hydroperoxide (tBH), menadione, methyl vinyl ketone] and the cytotoxicity of "chemical hypoxia" [i.e. KCN + iodoacetic acid] were greatest in air, intermediate in oxygen, and lowest in nitrogen. In contrast, the cytotoxicity of the alkylating agent N-dimethylnitrosamine was independent of oxygen concentration and the cytotoxicity of p-aminophenol was related directly to oxygen concentration. The mechanism of the oxygen dependence of chemical injury was investigated further, employing tBH as a model toxicant. tBH metabolism was oxygen independent in both PT and DT cells. Depletion of cellular protein sulfhydryl groups by tBH increased with increasing oxygen concentration and lipid peroxidation due to tBH was inhibited in nitrogen but was not different in air as compared with oxygen. Although these processes may contribute to the much lower toxicity in nitrogen as compared with oxygen, it does not explain the higher toxicity in air as compared with that in oxygen. Other processes that predominate at lower oxygen concentrations but that only produce injury if enough oxygen is present are likely to be responsible for the enhanced susceptibility of both PT and DT cells to oxidants in air as compared with oxygen.