The effect of pH on misonidazole-induced cell killing at normal and elevated temperatures and on radiosensitization by misonidazole at 37 degrees C was assessed in FSaIIC fibrosarcoma cells in vitro. At doses of 5-500 microM for 1 hr, misonidazole was 1.5- to 2-fold more toxic toward hypoxic versus euoxic cells at 37 degrees C and pH 7.40. At 42 degrees C and 43 degrees C at pH 7.40, a less than 2-fold increase in cytotoxicity was observed in both normally oxic and hypoxic cells as compared with 37 degrees C. At pH 6.45 and 37 degrees C, misonidazole was less cytotoxic toward both euoxic and hypoxic cells than at pH 7.40. Unexpectedly, exposure to misonidazole at 42 degrees C or 43 degrees C and pH 6.45 caused no significant increase in cytotoxicity over that attributable to hyperthermia alone. Similarly, the dose modifying effect of misonidazole on single radiation fractions in vitro was also reduced at pH 6.45 versus pH 7.40 (2.60 versus 2.40, p less than 0.01). In vivo, treatment of the FSaIIC tumor with misonidazole (1 g/kg) and/or local hyperthermia (43 degrees C for 30 min to the tumor-bearing limb) in conjunction with radiation (10, 20, or 30 Gy) yielded a radiation dose modifying factor for misonidazole of 1.32, for hyperthermia of 1.38, and for the combination of 2.06 (probably additive). Analysis of the cytotoxicity achieved by these treatments in Hoechst 33342 dye-selected tumor subpopulations demonstrated that, whereas radiation was more toxic toward bright (presumably euoxic) cells, misonidazole, hyperthermia, and the combination were significantly more toxic toward dim (presumably hypoxic) cells. The addition of both hyperthermia and misonidazole to radiation more than overcame the relative resistance of the dim subpopulation to 10 Gy. These results indicate that misonidazole is a reasonable drug for use with hyperthermia and radiation to increase killing of hypoxic cells, but the decrease in cytotoxicity and radiosensitizing abilities of this agent observed under acidotic conditions could reduce the effectiveness of this treatment.