Four enzymic components, coded for by the entD, entE, entF and entG genes, involved in the biosynthesis of enterochelin from 2,3-dihydroxybenzoate have been separated from cell extracts of mutant strains of Escherichia coli K-12. The starting material for fractionation of the E, F and G components was a cell extract of an entD mutant strain, which yielded the E, F and G enzymic components uncontaminated by a functional D component. The D component was isolated from cell extracts of an entE mutant strain. The conversion of 2,3-dihydroxybenzoate and L-serine into enterochelin is dependent on the presence of all four enzymic components. The E and F components were shown to catalyze ATP-pyrophosphate exchange reactions dependent on 2,3-dihydroxybenzoate and L-serine, respectively, whereas fractionated extracts of the entE and entF mutant strains lacked these reactions. These data provide firm evidence that the E and F components are involved in the initial activation of the substrates. The D and G components are necessary for subsequent and, as yet, undefinedd reactions.