We recently showed that severe hypoxia was not universally present adjacent to necrosis in human glioma xenografts and spheroids established from the M059K, M006, M006X, M006XLo and M010b cell lines. Using these glioma models, we wished to test whether oxygen serves as a regulator of cellular VEGF expression in situ. In situ hybridization (ISH) and immunohistochemistry (IHC) were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression in sections of glioma xenografts and spheroids in which hypoxic regions and regions with well-oxygenated necrosis were identified on contiguous sections by use of the hypoxia-specific marker, 3H-misonidazole. Independent validation of the presence of radiobiologically hypoxic cells in M006 xenografts was undertaken using the comet assay. Northern blotting analyses of monolayer cells demonstrated significant up-regulation of VEGF mRNA in the M006X line at oxygen concentrations of 6% and below. ISH analysis of VEGF mRNA showed unexpectedly strong staining for VEGF mRNA across the entire viable rim of M006X and M006XLo glioma spheroids. Similarly, in virtually all xenograft tumours of the M059K, M006 and M010b lines, VEGF ISH showed similar staining across all regions of healthy cells up to the border of necrosis. Only in one M006X tumour was there a suggestion of increased VEGF expression in cells adjacent to necrosis. IHC for VEGF showed good concordance with the ISH results. IHC analysis of the VEGF receptor flt-1 showed strong tumour cell staining in M006XLo glioma cells. In human glioma spheroids and xenograft tumours, regions of severe hypoxia do not correspond to areas of up-regulated VEGF expression; in fact, VEGF expression is quite uniform. Furthermore, this and our previous study demonstrate that levels of VEGF expression vary among sublines (M006, M006X and M006XLo) derived from a single human glioma specimen.