A series of 3,6-substituted 2,5-bis(1-aziridinyl)-1,4-benzoquinone derivatives was shown to alkylate calf thymus DNA and to form DNA interstrand cross-links. Alkylation and cross-link formation were enhanced after electrochemical reduction of the compounds and increased with lower pH in the pH range from 4.5 to 8.0. Reduction especially shifts the pH at which cross-linking and alkylation occurs to higher values, which are more physiologically relevant. This shift is probably caused by the increase in pKa value of the aziridine ring after reduction of the quinone moiety. The inactivation of single-stranded bacteriophage M13mp19 DNA to form phages in an E. coli host, by the 3,6-unsubstituted parent compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone (TW13) was dependent upon reduction and pH in a similar way as was alkylation. The compound in our series with the least bulky, 3,6-substituents, TW13, caused a high amount of cross-link formation. Compounds with methyl-substituted aziridine rings showed low cross-linking ability. Our results support the concept that the protonated reduced compound is the reactive species that alkylates DNA, and that steric factors play an important role in the reactivity towards DNA. A correlation is observed between the ability to induce DNA interstrand cross-links and inactivation of M13mp19 bacteriophage DNA. Cross-link formation was also demonstrated in E. coli K12 cells, where the compounds are reduced endogenously by bacterial reductases.