The sole biosynthetic route to S-adenosylmethionine, the primary biological alkylating agent, is catalysed by S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase). In Escherichia coli and Salmonella typhimurium numerous studies have located a structural gene (metK) for this enzyme at 63 min on the chromosomal map. We have now identified a second structural gene for S-adenosylmethionine synthetase in E. coli by DNA hybridization experiments with metK as the probe; we denote this gene as metX. The metX gene is located adjacent to metK with the gene order speA metK metX speC. The metK and metX genes are separated by approximately 0.8 kb. The metK and the metX genes are oriented convergently as indicated by DNA hybridization experiments using sequences from the 5' and 3' ends of metK. The metK gene product is detected immunochemically only in cells growing in minimal media, whereas the metX gene product is detected immunochemically in cells grown in rich media at all growth phases and in stationary phase in minimal media. Mutants in metK or metX were obtained by insertion of a kanamycin resistance element into the coding region of the cloned metK gene (metK::kan) followed by use of homologous recombination to disrupt the chromosomal metK or metX gene. The metK::kan mutant thus prepared does not grow on minimal media but does grow normally on rich media, while the corresponding metX::kan mutant does not grow on rich media although it grows normally on minimal media. These results indicate that metK expression is essential for growth of E. coli on minimal media and metX expression is essential for growth on rich media. Our results demonstrate that AdoMet synthetase has an essential cellular and/or metabolic function. Furthermore, the growth phenotypes, as well as immunochemical studies, demonstrate that the two genes that encode S-adenosylmethionine synthetase isozymes are differentially regulated. The mutations in metK and metX are highly unstable and readily yield kanamycin-resistant cells in which the chromosomal location of the kanamycin-resistance element has changed.