Menaquinone is an essential vitamin as an obligatory component of the electron transfer pathway in microorganisms. Menaquinone has been shown to be derived from chorismate by eight enzymes, designated MenA to -H in Escherichia coli. However, bioinformatic analyses of whole-genome sequences have suggested that some microorganisms, such as Helicobacter pylori and Campylobacter jejuni, which are known to cause gastric carcinoma and diarrhea, respectively, do not have orthologs of most of the men genes, although they synthesize menaquinone. The (13)C-labeling pattern of menaquinone purified from Streptomyces coelicolor A3(2) grown on [U-(13)C]glucose was quite different from that of E. coli, suggesting that an alternative pathway was operating in the strain. We searched for candidate genes participating in the alternative pathway by in silico screening, and the involvement of these genes in the pathway was confirmed by gene-disruption experiments. We also used mutagenesis to isolate mutants that required menaquinone for their growth and used these mutants as hosts for shotgun cloning experiments. Metabolites that accumulated in the culture broth of mutants were isolated and their structures were determined. Taking these results together, we deduced the outline of the alternative pathway, which branched at chorismate in a similar manner to the known pathway but then followed a completely different pathway. As humans and some useful intestinal bacteria, such as lactobacilli, lack the alternative pathway, it would be an attractive target for the development of chemotherapeutics.