1-Ethyl-1-nitrosourea (ENU) is a potent monofunctional ethylating agent that has been found to be mutagenic in a wide variety of mutagenicity tests system from viruses to mammalian germ cells. It also has been shown to induce tumors in various organs of mammals. ENU has been used only for research purposes. ENU possesses the dual action of ethylation and carbamoylation. The ethyl group can be transferred to nucleophilic sites of cellular constituents, and the carbonyl group can be transferred to an amino group of protein. ENU is able to produce significant levels of alkylation at oxygens, such as the O6 position of guanine and the O4 position of thymine of DNA. The molecular genetic data obtained from ENU-induced mutants on various species suggest that ENU produces mainly GC-AT transitions and, to a small extent, AT-GC, AT-CG, AT-TA, GC-CG and GC-TA base substitutions. This mutation spectrum of ENU is different from that of 1-methyl-1-nitrosourea, which mainly induces GC-AT transitions. ENU is a most potent mutagen in mouse germ cells, especially in stem-cell spermatogonia. It induces intragenic mutations with high frequency in male mouse germ cells. ENU has been established as a model compound for exploring the effects of chemical mutagenesis on mouse germ cells.