The antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine, TPZ, 1) gains medicinal activity through its ability to selectively damage DNA in the hypoxic cells found inside solid tumors. This occurs via one-electron enzymatic reduction of TPZ to yield an oxygen-sensitive drug radical (2) that leads to oxidatively generated DNA damage under hypoxic conditions. Two possible mechanisms have been considered to account for oxidatively generated DNA damage by TPZ. First, homolysis of the N-OH bond in 2 may yield the well-known DNA-damaging agent, hydroxyl radical. Alternatively, it has been suggested that elimination of water from 2 generates a benzotriazinyl radical (4) as the ultimate DNA-damaging species. In the studies described here, the TPZ analogue 3-methyl-1,2,4-benzotriazine 1,4-dioxide (5) was employed as a tool to probe the mechanism of DNA damage within this new class of antitumor drugs. Initially, it was demonstrated that 5 causes redox-activated, hypoxia-selective oxidation of DNA and small organic substrates in a manner that is completely analogous to TPZ. This suggests that 5 and TPZ damage DNA by the same chemical mechanism. Importantly, the methyl substituent in 5 provides a means for assessing whether the putative benzotriazinyl intermediate 7 is generated following one-electron reduction. Two complementary isotopic labeling experiments provide evidence against the formation of the benzotriazinyl radical intermediate. Rather, a mechanism involving the release of hydroxyl radical from the activated drug radical intermediates can explain the DNA-cleaving properties of this class of antitumor drug candidates.