The effects of insulin on tumor oxygenation, perfusion, oxygen consumption,and radiation sensitivity were studied on two different mouse tumor models (TLT, a liver tumor, and FSAII, a fibrosarcoma). Anesthetized mice were infused with insulin i.v. at a rate of 16 milliUnits/kg/min for 25 min. Local tumor oxygenation measurements were carried out using two independent techniques: electron paramagnetic resonance oximetry and a fiber-optic device (OxyLite). Two complementary techniques were also used to assess the blood flow inside the tumor: a laser Doppler system (OxyFlo) and contrast-enhanced magnetic resonance imaging. The oxygen consumption rate of tumor cells after in vivo insulin infusion was measured using high frequency electron paramagnetic resonance oximetry. To know if insulin was able to enhance radiation-induced tumor regrowth delay, tumor-bearing mice were treated with 16 Gy of 250 kV radiation dose after insulin infusion. We provide evidence that insulin increases the local pressure of oxygen of tumors (from 0-3 mm Hg to 8-11 mm Hg) as well as the tumor response to irradiation (increasing regrowth delay by a factor of 2.11). We found that the insulin-induced increase of tumor pressure of oxygen: (a) is not caused by an increase in the tumor blood flow, which is even decreased after insulin infusion; (b) is because of a decrease in the tumor cell oxygen consumption (in vivo insulin consumed oxygen three times slower than control cells); and (c) is inhibited by a nitric oxide (NO) synthase inhibitor, Nomega-nitro-L-arginine methyl ester, when injected i.p. at 15 micromol/kg(-1), 1 h before insulin infusion. We demonstrate by immunoblotting that the NO pathway involves a phosphorylation of endothelial NO synthase and showed a concomitant increase in the cyclic GMP tumor level. These findings provide unique insights into biological processes in tumors, new possible management for treating cancer patients, and raise major questions about the role of insulin secretion (fasting status and diabetes) in the clinical response of tumors to radiation therapy.