Four independent menaquinone (vitamin K2-deficient mutants of Escherichia coli, blocked in the conversion of o-succinylbenzoate (OSB) to 1,4-dihydroxy-2-naphthoate (DHNA), were found to represent two distinct classes. Enzymatic complementation was observed when a cell-free extract of one mutant was mixed with extracts of any of the remaining three mutants. The missing enzymes in the two classes were identified by in vitro complementation with preparations of OSB-coenzyme A (CoA) synthetase or DHNA synthase isolated from Mycobacterium phlei. Mutants lacking DHNA synthase (and therefore complementing with M. phlei DHNA synthase) were designated menB, and the mutant lacking OSB-CoA synthetase (and therefore complementing with M. phlei OSB-CoA synthetase) was designated menE. The menB mutants produced only the spirodilactone form of OSB when extracts were incubated with [2,3-14C2]OSB, ATP, and CoA; the OSB was unchanged on incubation with an extract from the menE mutant under these conditions. Experiments with strains lysogenized by a λ men transducing phage (λG68) and transduction studies with phage P1 indicated that the menB and menE genes form part of a cluster of four genes, controlling the early steps in menaquinone biosynthesis, located at 48.5 min in the E. coli linkage map. Evidence was obtained for the clockwise gene order gyrA....menC-E⃰ -B-D, where the asterisk denotes the uncertain position of menE relative to menC and menB. The transducing phage (λG68) contained functional menB, menC, and menE genes, but only part of the menD gene, and it was designated λ menCE⃰ B(D).