Longitudinal PO₂ profiles in the microvasculature of the rat mesentery were studied using a novel phosphorescence quenching microscopy technique that minimizes the accumulated photo-consumption of oxygen by the method. Intravascular oxygen tension (PO₂, mmHg) and vessel diameter (d, µm) were measured in mesenteric microvessels (N = 204) of 7 anesthetized rats (275 g). The excitation parameters were as follows: spot size 7x7 µm, 410 nm laser, and 100 curves at 11 pulses/s, with pulse parameters of 2 µs duration and 80 pJ/µm² energy density. The mean PO₂ (M ± SE) was 65.0 ± 1.4 mmHg (N = 78) for arterioles (d = 18.8 ± 0.7 µm), 62.1 ± 2.0 mmHg (N = 38) at the arteriolar end of capillaries (d = 7.8 ± 0.3 µm), and 52.0 ± 1.0 mmHg (N = 88) for venules (d = 22.5 ± 1.0 µm). There was no apparent dependence of PO₂ on d in arterioles and venules. There were also no significant deviations in PO₂ based on d (bin width 5 µm) from the general mean for both of these types of vessels. Results indicate that the primary site of oxygen delivery to tissue is located between the smallest arterioles and venules (change of 16.3 mmHg, p = 0.001). In conclusion, oxygen losses from mesenteric arterioles and venules are negligible, indicating low metabolic rates for both the vascular wall and the mesenteric tissue. Capillaries appear to be the primary site of oxygen delivery to the tissue in the mesenteric microcirculation. In light of the present results, previously reported data concerning oxygen consumption in the mesenteric microcirculation can be explained as artifacts of accumulated oxygen consumption due to the application of instrumentation having a large excitation area for PO₂ measurements in slow moving and stationary media.