Nicotinamide, an agent previously reported to reduce hypoxia and increase the irradiation response of experimental tumors, has been evaluated for its effect on the occurrence of acute hypoxia in the murine squamous cell tumor SCCVII. Treatment of C3H mice bearing 500-750-mg subcutaneous tumors with nicotinamide (1.0 mg/g intraperitoneally) 1 hour prior to irradiation resulted in an enhancement ratio of 1.3 (+/- 0.1). We assessed the effect of nicotinamide on the response of acutely hypoxic cells in vivo using a recently developed fluorescence-activated-cell sorting technique. This technique employs the in vivo pharmacokinetic and DNA binding properties of the bisbenzamide stain Hoechst 33342. The results clearly show that nicotinamide, at the doses used, reduces the amount of acute hypoxia in these SCCVII tumors. We confirmed these findings using a histological technique that facilitates the assessment of functional tumor vasculature at two instances in time. This method shows that nicotinamide reduces the number of vessels opening and closing over a 20-minute period from 10.3% to 2.0%. The identification of a compound that can modify the dynamic fluctuations in microregional oxygen delivery in tumors could have important implications for radiation therapy.