The primary structure of a multifunctional protein, the large alpha-subunit of the Escherichia coli fatty acid oxidation complex, was determined by sequencing the fadB region of the fadBA operon. The amino-terminal sequence of this protein had been established by Edman degradation. The transcription start site of the fadBA operon was located 42 nucleotides upstream of the initiator codon of the fadB gene by primer extension analysis. Sequences of -10 and -35 regions of the promoter responsible for interaction with RNA polymerase were found to be CACACT and TTTGCA, respectively. The location of the promoter of the fadBA operon was defined, and the transcription direction of this operon, from fadB to fadA, as previously proposed [Yang, S.-Y., et al. (1990) J. Biol. Chem. 265, 10424-10429], was corroborated. The multifunctional protein is composed of 729 amino acid residues and has a calculated Mr of 79,593. A putative NAD-binding beta alpha beta-fold necessary for L-3-hydroxyacyl-CoA dehydrogenase function was found in the central region of the fadB gene product. Sequence analyses suggest that the functional domains of the multifunctional protein are arranged in the order enoyl-CoA hydratase:L-3-hydroxyacyl-CoA dehydrogenase: delta 3-cis-delta 2-trans-enoyl-CoA isomerase and suggest that the genes of the E. coli multifunctional protein and rat peroxisomal trifunctional beta-oxidation enzyme evolved from a common ancestral gene.