To test the hypothesis that temporal variations in microvessel red cell flux cause unstable oxygen levels in tumor interstitium, extravascular oxygenation of R3230Ac mammary tumors grown in skin-fold window chambers was measured using recessed tip polarographic microelectrodes. Red cell fluxes in microvessels surrounding pO₂ measurement locations were measured using fluorescently labeled red cells. Temporal pO₂ instability was observed in all experiments. Median pO₂ was inversely related to radial distance from microvessels. Transient fluctuations above and below 10 mm Hg were consistently seen, except in one experiment near the oxygen diffusion distance limit (140 μm) where pO₂ fluctuations were <2 mm Hg and median pO₂ was <5 mm Hg. Vascular stasis was not seen in these experiments. These results show that fluctuations in red cell flux, as opposed to vascular stasis, can cause temporal variations in pO₂ that extend from perivascular regions to the maximum oxygen diffusion distance. (Cancer Res 2006; 66(4): 2219-23)