Expression of Escherichia coli open reading frame yfcX is shown to be required for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) formation from fatty acids in an E. coli fadB mutant. The open reading frame encodes a protein, YfcX, with significant similarity to the large subunit of multifunctional beta-oxidation enzymes. E. coli fadB strains modified to contain an inactivated copy of yfcX and to express a medium-chain-length synthase are unable to form PHA(MCL)s when grown in the presence of fatty acids. Plasmid-based expression of yfcX in the FadB(-) YfcX(-) PhaC(+) strain restores polymer formation. YfcX is shown to be a multifunctional enzyme that minimally encodes hydratase and dehydrogenase activities. The gene encoding YfcX is located downstream from yfcY, a gene encoding thiolase activity. Results of insertional inactivation studies and enzyme activity analyses suggest a role for yfcX in PHA monomer unit formation in recombinant E. coli fadB mutant strains. Further studies are required to determine the natural role of YfcX in the metabolism of E. coli.