Incubation of Ehrlich ascites tumor cells with misonidazole under aerobic conditions causes a stimulation of glucose consumption which is probably related to stimulation of hexose monophosphate shunt activity. Incubation of Ehrlich cells with 5 mM misonidazole under hypoxic conditions results in a time-dependent inhibition of glycolysis, seen when treated cells are washed and resuspended in fresh buffered saline or media. This inhibition does not appear to be related to breakdown and loss of pyridine nucleotides from the cells during misonidazole treatment, nor is it a consequence of cell death. Post-incubation in buffer containing cysteine or cysteamine restores cellular glycolytic activity to near control levels. Partial inhibition of glucose consumption after incubation with misonidazole also occurs with V79-379A, V79-171B, EMT6 and A549 cell lines. The extent of inhibition varies among the lines, but is accompanied by approximately a 50% reduction in intracellular non-protein thiol levels in all cases. Ehrlich cells incubated anaerobically with misonidazole lose their response to the uncoupler 2,4-dinitrophenol, which normally increases glucose consumption, and exhibit less ability to metabolize pyruvate. Cells incubated under anaerobic conditions but in the absence of misonidazole do not show these effects.