Carbogen (95% O 2 and 5% CO 2) has been used in preference to 100% oxygen (O 2) as a radiosensitizer, because it is believed that CO 2 blocks O 2-induced vasoconstriction. However, recent work suggests that both normal and tumour arterioles of dorsal flap window chambers exhibit the opposite: no vasoconstriction vs constriction for O 2 vs carbogen breathing respectively. We hypothesized that CO 2 content might cause vasoconstriction and investigated the effects of three O 2–CO 2 breathing mixtures on tumour arteriolar diameter (TAD) and blood flow (TBF). Fischer 344 rats with R3230Ac tumours transplanted into window chambers breathed either 1%, 5%, or 10% CO 2+ O 2. Intravital microscopy and laser Doppler flowmetry were used to measure TAD and TBF respectively. Animals breathing 1% CO 2 had increased mean arterial pressure (MAP), no change in heart rate (HR), transient reduction in TAD and no change in TBF. Rats breathing 5% CO 2 (carbogen) had transiently increased MAP, decreased HR, reduced TAD and a sustained 25% TBF decrease. Animals exposed to 10% CO 2 experienced a transient decrease in MAP, no HR change, reduced TAD and a 30–40% transient TBF decrease. The effects on MAP, HR, TAD and TBF were not CO 2 dose-dependent, suggesting that complex physiologic mechanisms are involved. Nevertheless, when≥ 5% CO 2 was breathed, there was clear vasoconstriction and TBF reduction in this model. This suggests that the effects of hypercarbic gases on TBF are site-dependent and that use of carbogen as a radiosensitizer may be counterproductive in certain situations.