The radiosensitizing and cytotoxic properties of Mitomycin C (MMC) was investigated in vivo using regrowth delay and tumor control assays. MMC significantly enhanced the radiation-induced growth delay when administered 15 min before irradiation; the slope of the dose response curve significantly increased and corresponded to a Dose Modifying Factor (DMF) of 1.9 (1.5-2.3; p less than 0.001). When MMC was given 4 hr after irradiation, the additional regrowth delay resulted in a parallel shift of the dose response curve, and MMC was not significantly dose modifying (DMF 1.3 (0.9-1.3); p less than 0.05). From isobologram analysis it was found that the preirradiation MMC schedule resulted in supra-additive responses, whereas MMC given after irradiation had an additive effect. The enhancement of radiation-induced tumor control was similarly found to peak when MMC was given 6 hr to 15 min prior to irradiation. At these intervals, the observed TCD50 for the combined treatments relative to radiation alone corresponded to Enhancement Ratios of 1.27 and 1.29, respectively (p less than 0.001). Longer intervals between the modalities reduced the enhancement, but the combined treatments were still significantly better than radiation alone (ER 1.12, 1.16 and 1.17; p less than 0.001). The significant enhancement of tumor control correlated with a substantial drug-induced cytotoxic effect toward hypoxic tumor cells, as determined by clamped TCD50 experiments. A single dose of MMC (3 mg/kg) was found to kill up to 97% of all hypoxic tumor cells.