Abstract C57BL male mice were injected with N-methyl- N-nitrosourea (MNUA) or N-ethyl- N-nitrosourea (ENUA) and the concentration of alkyl phosphotriesters in the DNA of lung, liver, brain, kidney, spleen and thymus determined from the extent of degradation induced in isolated DNA by alkali. The same total dose of reagent was given either as a single injection (i.p.) or by weekly injections carried out over 5–20 weeks. Methyl phosphotriesters induced in liver, lung and kidney by the single injection were lost with a half-life of about 7 days, in brain the loss was more rapid, t 1 2 = 2–3 days . During the multiple injections the observed t 1 2 was 16 days. Ethyl phosphotriesters formed in the DNA of lung, liver, kidney and brain were much more stable than the methyl derivatives, t 1 2 = 10–15 weeks . Phosphotriesters formed in the DNA of spleen and thymus disappeared very quickly after the single injection presumably as a result of dilution due to DNA replication. No accumulation of phosphotriesters occurred in the DNA of these tissues during the multiple injections. The general pattern of the results suggests that phosphotriesters are not excised by cellular repair systems.