After treatment with methylnitrosamine, samples of DNA were isolated from rat livers by a conventional phenol procedure and examined for the presence ofphosphotriesters. A method capable of detecting relatively small amounts of '4C-labelled phosphotriesters was developed and used to establish that these products account for 10-12% of the total methylation pattern found after treatment with this agent in vivo. The significance of the presence of phosphotriesters in DNA is discussed.