We previously reported that the arteriolar input in window chamber tumours is limited in number and is constrained to enter the tumour from one surface, and that the pO 2 of tumour arterioles is lower than in comparable arterioles of normal tissues. On average, the vascular pO 2 in vessels of the upper surface of these tumours is lower than the pO 2 of vessels on the fascial side, suggesting that there may be steep vascular longitudinal gradients (defined as the decline in vascular pO 2 along the afferent path of blood flow) that contribute to vascular hypoxia on the upper surface of the tumours. However, we have not previously measured tissue pO 2 on both surfaces of these chambers in the same tumour. In this report, we investigated the hypothesis that the anatomical constraint of arteriolar supply from one side of the tumour results in longitudinal gradients in pO 2 sufficient in magnitude to create vascular hypoxia in tumours grown in dorsal flap window chambers. Fischer-344 rats had dorsal flap window chambers implanted in the skin fold with simultaneous transplantation of the R3230AC tumour. Tumours were studied at 9–11 days after transplantation, at a diameter of 3–4 mm; the tissue thickness was 200 μm. For magnetic resonance microscopic imaging, gadolinium DTPA bovine serum albumin (BSA-DTPA-Gd) complex was injected iv, followed by fixation in 10% formalin and removal from the animal. The sample was imaged at 9.4 T, yielding voxel sizes of 40 μm. Intravital microscopy was used to visualize the position and number of arterioles entering window chamber tumour preparations. Phosphorescence life time imaging (PLI) was used to measure vascular pO 2. Blue and green light excitations of the upper and lower surfaces of window chambers were made (penetration depth of light~ 50 vs> 200 μm respectively). Arteriolar input into window chamber tumours was limited to 1 or 2 vessels, and appeared to be constrained to the fascial surface upon which the tumour grows. PLI of the tumour surface indicated greater hypoxia with blue compared with green light excitation (P< 0.03 for 10th and 25th percentiles and for per cent pixels< 10 mmHg). In contrast, illumination of the fascial surface with blue light indicated less hypoxia compared with illumination of the tumour surface (P< 0.05 for 10th and 25th percentiles and for per cent pixels< 10 mmHg). There was no significant difference in pO 2 distributions for blue and green light excitation from the fascial surface nor for green light excitation when viewed from either surface. The PLI data demonstrates that the upper surface of the tumour is more hypoxic because blue light excitation yields lower pO 2 values than green light excitation. This is further verified in the subset of chambers in which blue light excitation of the fascial surface showed higher pO 2 distributions compared with the tumour surface. These results suggest that there are steep longitudinal gradients in vascular pO 2 in this tumour model that are created by the limited number and orientation of the arterioles. This contributes to tumour hypoxia. Arteriolar supply is often limited in other tumours as well, suggesting that this may represent another cause for tumour hypoxia. This report is the first direct demonstration that longitudinal oxygen gradients actually lead to hypoxia in tumours.