The aim of this study was to evaluate the effect of carbogen breathing on the physiological profile of human glioma xenografts. Near infrared spectroscopy was used to investigate changes in oxy- and deoxyhemoglobin concentrations in tumor blood. Oxygen tension changes in tumor tissue were evaluated by 19F-MR relaxometry, using perfluoro-15-crown-5-ether, and modifications of tumor blood perfusion (TBP) were analyzed by fast dynamic 1H-MR imaging of Gd-DTPA uptake. Finally, changes of the bioenergetic status and pH of tumor cells were analyzed by 31P-MRS. After 5 to 8 min of carbogen breathing, the average oxygen tension increase in tumor tissue was 4.6 ± 1.3 mm Hg, which is in agreement with an increase of the oxyhemoglobin concentration in tumor blood ($Δ$[O2Hb] = 9.2 ± 3 $μ$M). However, simultaneously the TBP was reduced, the bioenergetic status was diminished, and pH was decreased. As 100% O2 breathing alone did not result in a detectable increase of oxyhemoglobin in tumor blood, the increase of the tumor oxygenation by carbogen appears to be mediated by its CO2 content. This component may cause a nutrient-limited decrease of oxidative energy metabolism, indirectly via a steal-effect and/or by inhibition of the glycolytic rate resulting from tissue acidification.