CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) becomes, upon bioactivation, a difunctional alkylating agent. It can be up to a 100,000-fold more cytotoxic in cells that are able to bioactivate it than in those that cannot. This increase in cytotoxicity is much greater than would be predicted from the conversion of a monofunctional alkylating agent to a difunctional one. We now show that the interstrand crosslink formed in the DNA of CB 1954-sensitive cells has some unusual properties. In Walker cells, which are able to activate CB 1954, the interstrand crosslink is the major adduct and can constitute up to 70% of the total adducts. These crosslinks are only poorly excised, as are those produced in V79 cells (which are themselves unable to activate CB 1954) by co-culturing them with Walker cells. Also, CB 1954 is approximately 10-fold more reactive toward the DNA of Walker cells than V79 cells. These observations may explain the extent of the increase in cytotoxicity accompanying the bioactivation of CB 1954.