DNA is considered to be an important target for the antitumor and toxic properties of the Chloroethylnitrosoureas. Since the main target for their dose-limiting toxicity and the antileukemic efficacy is believed to be the bone marrow, we have compared the formation and subsequent removal of DNA-DNA interstrand cross-links in the bone marrow of rats which had received a single i.p. injection (100 ^mol/kg) of four Chloroethylnitrosoureas. The kinetics of cross-link removal was identical for chlorozotocin, which is known to have low chemical carbamoylating activity, to that of 1,3-bis(2-chloroethyl)-1-nitrosourea, a drug with a rela tively high carbamoylating capacity. The differential bone marrow toxicity exhibited by these two agents could not, therefore, be explained by a carbamoylation-mediated difference in the rate and extent of DNA-DNA interstrand cross-link removal. The peak level and overall magnitude of cross-links were, however, found to vary considerably with the chemical structure of the ana logues. Both 1-(2-chloroethyl)-1-nitroso-3-(methylene-carboxamido)urea and 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea, were much more effective in inducing interstrand cross-links than 1,3-bis(2-chloroethyl)-1-nitrosourea or chlorozotocin. This differential cross-linking did not, however, parallel the singledose acute toxicity of these agents but reflected to a greater extent differences in their antileukemic activity. Considering the widely differing biological properties of this class of compounds, the measurement of DNA-DNA interstrand cross-linking in vivo might prove relevant in the evaluation of novel nitrosoureas.