FabB (β- ketoacyl-acyl carrier protein synthase Ⅰ) and FabF(β- ketoacyl-acyl carrier protein synthase Ⅱ) are two key enzymes of fatty acid biosynthesis in E.coli. The Gram-positive pathogenic bacterium Enterococcus faecalis has a fatty acid composition very similar to that of E.coli. Bioinformatic analysis reveals that though E. faecalis has two fabF homologues, there is no recognizable fabB homologue in the genome of E. faecalis. Two fabF homologues (fabF1 and fabF2) were amplified by using E. faecalis V583 genomitic DNA as template, and two plasmids, pHW13 (fabF1) and pHW14 (fabF2), were constructed. The results of experiments in vivo and in vitro have shown that fabF1 gene could complement E. coli fabB mutation and FabF1 possessed β- ketoacyl-acyl carrier protein synthase Ⅰ(FabB) activity, while fabF2 gene could complement E. coli fabF mutation and FabF2 had β- ketoacyl-acyl carrier protein synthase Ⅱ(FabF) activity. Meanwhile the data also shown that FabF2 possessed partial function of β-ketoacyl-acyl carrier protein synthase Ⅰ(FabB), and it could make E. coli fabB mutation synthesized low amount of unsaturated fatty acid. From these data it is clear that FabF species enzymes could have activity of β- ketoacyl-acyl carrier protein synthase Ⅰ(FabB).