In prokaryotes, ubiquinone and menaquinone(MK) are obligatory components of the electron-transfer pathway. Escherichia coli, which is a facultative anaerobe, utilizes ubiquinone under aerobic conditions, but uses MK 8 when it is grown anaerobically. By contrast, many Gram-positive aerobes such as Bacillus subtilis contain only MKs. MK biosynthesis is therefore essential for the survival of these strains. MK is biosynthesized from chorismate by seven enzymes (MenA-MenH) in E. Coli. However, a bioinformatic analysis of whole genome sequences has suggested that some microorganisms, including pathogenic species such as Helicobacter pylori and Campylobacter jejuni, do not have orthologs of the men genes, even though they synteesize MK. Here, we investigated this novel pathway in a nonpathogenic Streptomyces coelicolor A3(2) grown on [U-^<13>C]glucose was quite different from that of E. coli. We searched for candidate genes participating in the pathway by in silico screening, and the involmement 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 the mutants were isolated and their structures were determined. Taking these results together, we deduced the outline of the alternative pathway, which formed chorismate in a similar manner to the known pathway, but then branched to follow a completely different pathway. Since humans and some useful intestinal bacteria utilize the different pathway, this new pathway would be an attractive target for the development of chemotherapeutics.