The toxicity of 5-thio-D-glucose (5TG) to mammalian cells in culture has been studied with respect to oxygen tension, concentration, and temperature. At 37 degrees C and at 5 mM concentration of the drug in normal growth medium, survival is 10(-3) for 4-hr exposure to 5 ppm O2; this increases to 0.5 for 24-hr exposure to 200 ppm O2. The relationship between survival and oxygen tension is nonlinear with the greatest change occurring between 50 and 100 ppm. The drug is essentially nontoxic to aerated cells. Drug toxicity increases with concentration up to about 5 mM at which point a plateau is reached. The effect of elevated temperature is to reduce the time required to obtain a specific level of survival, but temperatures as high as 42 degrees C had only a slight effect on drug toxicity for oxygen tensions higher than 100 ppm. The effect of D-glucose on the toxicity of 5TG was studied, and an inverse relationship was established. At D-glucose concentrations greater than 20 mM the toxicity of 5TG was nullified regardless of oxygen tension or 5TG concentration.