5-(Aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is the active form of CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). This hydroxylamine is formed by the bioreduction of CB 1954 by the enzyme DT diaphorase and accounts for the highly selective cytotoxicity of this compound. The reason why the hydroxylamine derivative is so cytotoxic is that, in contrast to CB 1954, it can react difunctionally as characterized by the formation of DNA-DNA interstrand crosslinks in cells treated by this agent. However, although the 4-hydroxylamine compound can produce these crosslinks in cells it cannot crosslink naked DNA (Knox et al., Biochem Pharmacol 37: 4661-4669, 1988). We show here that 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide can become a species capable of binding to DNA and producing interstrand crosslinks, by a direct, non-enzymatic reaction with either acetyl coenzyme A, butyl and propyl coenzyme A or S-acetylthiocholine. Coenzyme A itself cannot produce these effects. The major product of the reaction between the 4-hydroxylamine and thioesters was identified as 4-amino-5-(aziridin-1-yl)-2-nitrobenzamide. However, this compound is not capable of producing the above effects and the major DNA reactive species was a minor product of the reaction. It is proposed that the ultimate, DNA reactive, derivative of CB 1954 is 4-(N-acetoxy)-5-(aziridin-1-yl)-2-nitrobenzamide.