FabB and FabF are two key long-chain 3-ketoacyl-ACP synthase in Escherichia coli. In addition to participating synthesis of long acyl chains, FabB is a key enzyme responsible for a condensation reaction in de novo unsaturated fatty acid synthesis and to form palmitoleoyl-ACP. While the FabF was found to be required for the elongation of cis-9-hexadecenoyl-ACP to cis-11-octadecenoyl-ACP and not involved in de novo unsaturated fatty acid synthesis. It has been reported previously that FabF homologues were found to have both KAS Ⅰ and Ⅱ activity just like FabB and FabF of E. coli in Enterococcus faecalis, Lactococcus lactis, Clostridium acetobutylicium and Ralstonia solanacearum. To test if this phenomenon is prevalent, we have carried out functional identification of five fabF homologous genes, Bacillus subtilis BsfabF, Sinorhizobium meliloti SmfabF, Vibrio cholera VcfabF, Pseudomonas aeruginosa PafabF1 and PafabF2. Our data revealed that five FabF homologues all have the long-chain 3-ketoacyl-ACP synthase activities in vitro. Analysis of phospholipid compositions show that SmfabF, VcfabF, PafabF1 and PafabF2 possessed 3-ketoacyl-ACP synthase Ⅱ (FabF) activity when complemented the E. coli fabF mutation CL28. The results of genetic complementation and thin-layer chromatographic analysis show that only PafabF2 gene could complement E. coli fabB mutation and PaFabF2 possessed partial function of 3-ketoacyl-ACP synthaseⅠ(FabB). These results demonstrated that not all FabF homologues have dual activity of KAS Ⅰ and KAS Ⅱ.