Escherichia coli b-ketoacyl synthases (KAS) I and II carry out the elongation steps in fatty acid synthesis. Analyses using the cross-linker BS₃ [bis(sulphosuccinimidyl) suberate] and surface-enhanced laser desorption/ionization–time-of-flight MS disclosed only monomeric and dimeric forms of KAS II, whereas KAS I also forms higher multimers. The binding affinities for KAS I and KAS II to C₁₄-acyl carrier protein (ACP) as well as for C₁₄-ACP to KAS I and KAS II were determined. KAS I is sensitive to the ACP released during the transfer reaction, with 50% inhibition at 0.17 µM ACP close to the physiological concentration of ACP (0.13 µM). KAS I and II also differ in carrying out the decarboxylation step of the elongation reaction.