Hypoxia is known to be an important prognostic marker in many human cancers. We report the use of two oxygen measurement techniques in human brain tumors and compare these data with semiquantitative histological end points. Oxygenation was measured using the Eppendorf needle electrode and/or EF5 binding in 28 brain tumors. These data were compared with necrosis, mitosis, and endothelial proliferation. In some tumors, absolute EF5 binding was converted to tissue pO(2) based on in vitro calibrations. Eppendorf electrode readings could not be used to identify WHO grade 1/2 versus WHO grade 3/4 tumors, they could not differentiate grade 3 versus grade 4 glial-derived neoplasms, nor did they correlate with necrosis or endothelial proliferation scores. EF5 binding increased as the tumor grade increased and was significantly associated with necrosis and endothelial proliferation. There was no statistically significant correlation between the two hypoxia detection techniques, although both methods indicated similar absolute ranges of tissue pO(2). There was substantial inter- and intratumoral heterogeneity of EF5 binding in WHO grade 4 glial neoplasms. The majority of cells in glial-derived tumor had levels of hypoxia that were mild to moderate (defined herein as 10% to 0.5% pO(2)) rather than severe (defined as approximately 0.1% pO(2)). Immunohistochemical detection of EF5 binding tracks histological parameters in adult brain tumors, with increased binding associated with increasing necrosis and endothelial proliferation. The proportion of moderately to severely hypoxic cells is relatively low, even in the high-grade tumors. Human brain tumors are dominated by oxic to moderately hypoxic cells.