The purpose of this study was to test the hypothesis that longitudinal O(2) gradients in tumor affect response to manipulation of oxygenation. Previously we showed that pO(2) is higher on the fascial than the tumor surface of the R3230Ac rat mammary carcinoma when growing in a dorsal skin-fold window chamber, reflecting a longitudinal oxygen gradient. Magnetic resonance angiography verified prior results: the fascial surface has arterioles and higher vascular density than tumor; and the tumor surface has no arterioles. Phosphorescence lifetime imaging was used to measure each surface hypoxic percentage (HP; percentage of pixels < 10 mm Hg) before and after administration of mannitol or glucose (1 g/kg, i.v.) followed by O(2) breathing. The fascial surface had a smaller HP (median = 2.72%) than tumor (median = 27.94%; P = 0.0002) at baseline. HP on the fascial surface was positively correlated with HP on the tumor surface (P = 0.0067). HP decreased on the fascial surface after either sugar + O(2) (mannitol P = 0.03; glucose P = 0.06; combined P = 0.002), but HP did not change on the tumor surface. Therefore, the tumor surface is refractory to improvement in pO(2) with this method. Additional refinements may be needed to improve pO(2) of analogous regions in larger tumors; mechanism-driven suggestions are provided.