The Escherichia coli gene cluster encoding enzymatic activities responsible for the synthesis and activation of 2,3-dihydroxybenzoic acid in the formation of the catechol siderophore enterobactin was localized to a 4.2-kilobase chromosomal DNA fragment. Analysis of various subclones and transposon insertion mutations confirmed the previously suggested gene order as entEBG(AC) and provided evidence to suggest that these genes are organized as three independent transcriptional units, composed of entE, entBG, and entAC, with the entBG mRNA transcribed in a clockwise direction. Plasmid-specific protein expression in E. coli minicells identified EntE and EntB as 58,000- and 32,500-dalton proteins, respectively, while no protein corresponding to EntG was detected. The EntA and EntC enzymatic activities could not be separated by genetic or molecular studies. A small DNA fragment encoding both activities expressed a single 26,000-dalton polypeptide, suggesting that this protein is a multifunctional enzyme catalyzing two nonsequential reactions in the biosynthetic pathway. A protein of approximately 15,000 daltons appears to be encoded by the chromosomal region adjacent to the entAC gene, but no known function in enterobactin biosynthesis or transport can yet be ascribed to this polypeptide.