Aziridinomitosenes are a class of compounds related to mitomycin C, an anti-cancer agent in current clinical use. We recently reported that a C-6/C-7 unsubstituted aziridinomitosene was found to form DNA interstrand crosslinks without the reductive activation required by mitomycin C, while retaining the same 5'-d(CG) sequence specificity. Herein we detail our efforts to verify a proposed mechanism for this previously unobserved event. Specifically, it is proposed that DNA monoalkylation occurs first at the aziridine carbon (C-1), followed by addition of solvent or another nucleophile to the quinone ring at C-6 or C-7, and subsequent crosslink formation at C-10. The hypothesis stems from the observations that 1) aziridinomitosenes are known to rapidly monoalkylate DNA, 2) the yield of interstrand crosslinks increases with time, and 3) the C-10 carbamate is essential for crosslink formation. To test this hypothesis, we have synthesized aziridinomitosene analogs with varying substitution patterns at the four potential electrophilic sites (C-1, C-6, C-7, and C-10). In order to gain insight into the roles these sites play in the proposed DNA crosslink-forming mechanism, the effects of the substitution patterns on the solvolytic, physical, and DNA binding properties of the aziridinomitosenes are being studied. Understanding the structural features and physical characteristics of aziridinomitosenes that form DNA crosslinks may lead to the enhancement and control of their formation.