Acyl-CoA analogs have been used in a relatively small number of enzyme structure-function studies, where the analogs can clarify roles of catalytic residues. These acyl-CoA analogs have the relatively labile thioester substituted with esters/amides/ketones to examine acyl-transfer catalysis, or the acyl-thioester is substituted by acyl-enolate isosteres to examine carbon-carbon bond forming enzymes. The thioester bond of malonyl-CoA is considered to be especially labile compared to acetyl-CoA. Thus, stable malonyl-thioester analogs are desired for use in structure-function studies. Other uses for stable malonyl-CoA analogs include off-loading polyketide synthase metabolic intermediates, or as probes of enzymes like carboxymethylproline synthase, where the analogs can participate as slow substrates. To determine suitability of stable acetyl/malonyl-thioester analogs in fatty acid and polyketide synthase structure-function studies or as inhibitor warheads, we report alternative syntheses of acetyl-oxa(dethia)CoA/acetyl-aza(dethia)CoA (1/2), malonyl-oxa(dethia)CoA/malonyl-aza(dethia)CoA (3/4) and their reactivity with Escherichia coli FabH, a ketosynthase (KS) initiating fatty acid biosynthesis, Figure . We also determine the inhibition constants of 1-4, oxa(dethia)CoA (5) and aza(dethia)CoA (6) against FabH using a continuous spectrophotometric assay applicable to other KS.