Nicotinamide has been shown to sensitize tumors to radiation in preference to normal tissues. We have extended our studies to examine the mechanism responsible for this radiosensitization, using the EMT6 tumor model. Our results confirm that nicotinamide (1000 mg/kg) significantly enhances the radiation damage in this tumor when given as a single intraperitoneal injection 90 min before irradiation. The data also show that nicotinamide does not directly sensitize hypoxic cells to radiation either in vitro or in vivo. Excising tumors immediately after irradiation and exposing them to nicotinamide (7 mM) for 24 h similarly failed to increase the radiation damage, implying that nicotinamide does not inhibit the repair of radiation-induced potentially lethal damage. Nicotinamide did, however, produce a decrease in the binding of [14C]-misonidazole in tumors, consistent with a reduction in the degree of tumor hypoxia. There was also an increase in mean tumor cell fluorescence of Hoechst 33342 in nicotinamide-treated mice compared to that of controls, suggesting that the increase in tumor oxygenation was probably a consequence of an increase in tumor blood perfusion.