In contrast to alkylating agents such as ethyl methanesulphonate which can induce mutation after treatment of free phage particles, nitrosamides induce mutations only when phage are treated intracellularly during infection of the host. The basis of this intracellular dependence is not currently understood. In this study the mutational specificity of nitrosomethylurea (NMU) in bacteriophage T4 was investigated by measuring the reversion of well-characterized mutants in the rII genes. While no mutation was produced after in vitro treatments of free phage, in vivo treatments strongly induced G:C leads to A:T transitions and substantially induced A:T leads to G:C transitions. Transversions and frameshift mutations were rarely induced. Although methyl methanesulfonate mutagenesis of T4 depends upon a phage-encoded error-prone repair system, NMU-induced mutagenesis is independent of this repair system. Similarities in mutagen specificity of nitrosamides and differences in DNA metabolism in T4 when compared to its host, Escherichia coli, suggest that T4 is well-suited for the study of mechanisms of nitrosamide mutagenesis.