The penetration of anticancer agents into tumor tissue has recently attracted considerable attention. This study examines the effect of carbogen breathing on the antitumor activity of tirapazamine combined with radiation. Our hypothesis is based on the observation that the diffusion of tirapazamine through tissue is dependent on oxygen tension. We postulated that carbogen breathing might enhance the ability of tirapazamine to diffuse to hypoxic cells located distal to functional blood vessels in tumors. We first determined that carbogen breathing caused no significant change in the pharmacokinetics of tirapazamine, suggesting that any effect of carbogen breathing on the activity of tirapazamine is not attributable to modulation of pharmacokinetics. Cell survival in SCCVII and SiHa tumors after 10 Gy X rays alone was similar. However, when tirapazamine was administered 30 min after radiation treatment under air-breathing conditions, cell killing was greater in SCCVII tumors compared to SiHa tumors. Carbogen breathing during the exposure to tirapazamine did not change the cell survival in SCCVII tumors, but it enhanced cell killing in the SiHa tumors. Interestingly, carbogen breathing during radiation treatment produced greater cell killing in the SiHa tumors than in the SCCVII tumors. The vascular architecture and type of hypoxia in the two tumors probably underlie the differences in the responses of the two tumors. These findings suggest that the effectiveness of tirapazamine and other hypoxic cytotoxins may be dependent on tumor type.