The anaerobic unsaturated fatty acid synthetic pathway of Escherichia coli requires two specialized proteins, FabA and FabB. However, the fabA and fabB genes are found only in the Gram-negative alpha- and gamma-proteobacteria, and thus other anaerobic bacteria must synthesize these acids using different enzymes. We report that the Gram-positive bacterium Enterococcus faecalis encodes a protein, annotated as FabZ1, that functionally replaces the E. coli FabA protein, although the sequence of this protein aligns much more closely with E. coli FabZ, a protein that plays no specific role in unsaturated fatty acid synthesis. Therefore E. faecalis FabZ1 is a bifunctional dehydratase/isomerase, an enzyme activity heretofore confined to a group of Gram-negative bacteria. The FabZ2 protein is unable to replace the function of E. coli FabZ, although FabZ2, a second E. faecalis FabZ homologue, has this ability. Moreover, an E. faecalis FabF homologue (FabF1) was found to replace the function of E. coli FabB, whereas a second FabF homologue was inactive. From these data it is clear that bacterial fatty acid biosynthetic pathways cannot be deduced solely by sequence comparisons.