Abstract Preliminary studies in vitro using bacteriophage T7-DNA have shown that breaks formed in the DNA on the alkaline hydrolysis of apurinic sites and phosphotriesters can be distinguished from each other by measuring the extent of degradation of the DNA immediately after adding NaOH to 0.1 M and after incubating for 1 h in 0.5 M NaOH. This method has then been applied to the study of the formation and stability of phosphotriesters in vivo. Methyl phosphotriesters formed in liver DNA following injection of mice with N-methyl- N-nitrosourea (MNUA) disappear with time (50% in 4–5 days). The concentration of ethyl phosphotriesters in liver DNA formed by injecting mice with N-ethyl- N-nitrosourea (ENUA) does not appear to decrease with time. Results of experiments on injecting methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and dimethyl sulphate (DMS) are also reported. The method described does not require the use of radioactively labelled reagents.