OBJECTIVE Although nicotinamide administration has increased the radiosensitivity of experimental tumors, there is a scarcity of data detailing the underlying physiological mechanisms. The current study presents a method for quantifying both microregional distributions of intravascular HbO2 saturations and the presence or absence of blood flow in adjacent frozen tumor sections. METHODS Two murine tumor cell lines, KHT and SCCVII, were implanted and quick-frozen without the use of anesthetics. Nicotinamide was administered IP 1 h prior to freezing, and a fluorescent dye that preferentially stains cells adjacent to blood vessels was injected i.v. 1 min prior to freezing. To visualize the presence or absence of blood flow, six micron sections were first cut using a cryostat. The remaining frozen tumor block was then analyzed cryospectrophotometrically to determine intravascular HbO2 levels. RESULTS While KHT HbO2 levels increased somewhat predictably following nicotinamide, the response in SCCVII tumors varied with distance from the tumor surface. Near the periphery, SCCVII HbO2 levels increased, but nearer the tumor center, HbO2 levels actually decreased. Perfused blood vessels were uniformly distributed throughout the tumor volume except in regions of necrosis. Even vessels containing no measurable oxygen remained perfused, as evidenced by the presence of the fluorescent marker. CONCLUSIONS These results demonstrate that nicotinamide raises intravascular HbO2 saturations in both KHT and SCCVII tumors. This increase in oxygen delivery is not evenly distributed throughout the tumor volume in spite of a uniform distribution of perfused blood vessels. Blood flow in a substantial proportion of these vessels is most likely not sufficiently rapid to serve a functional purpose in terms of oxygen supply to the surrounding tumor tissue.