Using an acyl-acyl carrier protein (ACP) as a starter unit, type II polyketide synthases (PKSs) generate a wide range of polyketide products by successive decarboxylative condensations with the two-carbon donor malonyl (ACP). In vitro experiments have demonstrated that polyketide biosynthesis in reconstituted PKS systems requires the fatty acid synthase (FAS) enzyme malonyl CoA:ACP acyltransferase (FabD) from streptomycetes. It has also been shown that holo-ACPs from a type II PKS can catalyze self-malonylation in the presence of malonyl CoA and negate this FabD requirement. The relative roles of FabD and ACP self-malonylation in PKS biosynthesis in vivo are still not known.We have examined the ACP specificity of the Streptomyces glaucescens FabD and shown that it reacts specifically with monomeric forms of ACP, with comparable k cat /K m values for ACPs from both type II PKS and FAS systems. Incubations of tetracenomycin ACP (TcmM) with the Escherichia coli FAS ACP (AcpP) unexpectedly revealed that, in addition to the self-malonylation process, TcmM can catalyze the malonylation of AcpP. The k cat /K M value for the TcmM-catalyzed malonylation of S. glaucescens FAS ACP is two orders of magnitude smaller than that observed for the FabD-catalyzed process.The ability of a PKS ACP to catalyze malonylation of a FAS ACP is a surprising finding and demonstrates for the first time that PKS ACPs and FabD can catalyze the same reaction. The differences in the catalytic efficiency of these two proteins rationalizes in vitro observations that FabD-independent polyketide biosynthesis proceeds only at high concentrations of a PKS ACP.