Aberrant architecture of the tumor vasculature and temporal fluctuations in blood flow can result in tumor hypoxia. The aim of this study was to classify tumor hypoxia based on distance to blood vessels, and to characterize its biologic significance by determining levels of nonprotein sulfhydryls (NPSH) in hypoxic regions located proximally and distally to tumor blood vessels.A dual fluorescence method was developed for the spatial colocalization of the vasculature and hypoxia in frozen sections from SiHa cervical carcinoma xenografts. A parallel section was stained with the sulfhydryl stain mercury orange. Composite fluorescence images were generated by imaging and tiling individual fields of view into 2D image arrays. Image arithmetic techniques were combined with feature-based image segmentation to characterize expression of NPSH as a function of the hypoxic tumor microenvironment.NPSH levels were higher in hypoxic areas of the SiHa xenografts (15.1 +/- 0.5 vs. 13.5 +/- 0.5 integrated optical density [IOD], p0.03). When tumor hypoxia was classified by distance to the nearest visible blood vessel, significantly higher NPSH levels were found in hypoxic regions close to blood vessels than in regions at a distance from blood vessels.The results of this study indicate differential expression of NPSH levels in regions of hypoxia that are proximal or distal to blood vessels in SiHa tumors.