The rates of chemical degradation of chloroethylnitrosoureas in serum are significantly higher than in aqueous buffer at the same pH and temperature. This rate enhancement is shown to be produced by a non-specific protein mediated chemical reaction that involves the formation of a protein-chloroethylnitrosourea (CENU) complex. Purified human serum albumin catalyzed reactions have been studied and Vm- and Km-values obtained for 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU). The rate of 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU) decomposition is not enhanced by serum proteins. Formation of a protein-BCNU complex can be inhibited by salicylic acid and dodecanoic acid, two compounds that have a high albumin binding affinity. Reaction product analysis indicates that the only BCNU reaction catalyzed by albumin is conversion to active 2-chloroethylazohydroxide and 2-chloroethylisocyanate intermediates. Formation of these reactive species at the protein surface leads to a high proportion of covalent bond formation to the protein. These results emphasize the complex and structure specific factors that may affect the biodistribution, antitumor activity and toxicity of members of the chloroethylnitrosourea class of cancer chemotherapeutic agents.