The mechanism by which a benzotriazine 1,4-dioxide class of anticancer drugs produce oxidizing radicals following their one-electron reduction has been investigated using tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide, 1) and its 6-methoxy (6), 7-dimethylamino M, and 8-methyl (8) analogues. By measuring the changes in absorption With pH, we found that the radical anions undergo protonation with radical pK(a) values of 6,19 +/- 0.05, 6.10 +/- 0.03, 6.45 +/- 0.04, and 6.60 +/- 0.04, respectively. The one-electron reduced species underwent a first-order reaction, with increased rate constants from 112 +/- 23 s(-1) for 1 to 777 +/- 12 s(-1)(6), 1120 +/- 29 s(-1) (7), and 825 +/- 89 s(-1) (8) at pH 7 No overall change in conductance was observed following the one-electron reduction of 6, and 8 at pH 4,5, consistent With the protonation of the radical anions, but a loss in conductance was seen for one-electron reduced 7 because of further protonation of the initially formed radical. This is assigned to the protonation of the dimethylamino group of the radical species, which has a pK(a) of 8.8 +/- 0.3. All conductance changes take place on a time-scale shorter than those of the above first-order reactions, which are not associated With the formation or loss of charged species. The absorption spectra present at the end of the unimolecular reactions were found to be similar to those formed immediately upon the one-electron oxidation of the respective substituted 3-amino-1,2,4-benzotriazine 1-oxides, and it is suggested that common benzotriazinyl radicals are formed by both routes. All these intermediate radicals underwent dismutation to produce final spectra matched by equal contributions of the parent compound and their respective substituted 3-amino-1,2,4-benzotriazine 1-oxides. By establishing redox equilibria between the intermediate radicals formed on the one-electron oxidation of the respective 3-amino-1,2,4-benzotriazine 1-oxides of the compounds and reference compounds, we found the one-electron reduction potential of the oxidizing radicals to range from 0.94 to 1.31 V. The benzotriazinyl radical of tirapazamine was found to oxidize dGMP and 2-deoxyribose with rate constants of (1.4 +/- 0.2) x 10(8) M-1 s(-1) and (3.7 +/- 0.5) x 10(6) M-1 s(-1), respectively.