In a previous study of fluoroacetyl-CoA hydrolase, the acetyl-CoA oxa/aza analogs were generated and compared with the corresponding fluorinated analogs as substrates. In those studies, the aza analogs were completely stable, whereas the oxa analogs were substrates at very low rates. In our study, we found that the oxa analogs undergo minor hydrolysis. With fluoroacetyl-CoA hydrolase, the fluoroacetyl-CoA Km is 8 µM, while the Kd for fluoroacetyl-oxa(dethia)CoA is 90 µM and fluoroacetyl-aza(dethia)CoA is 1700 µM, indicating large decreases in affinity for the oxa/aza substrate analogs. In our case, an assay that directly monitors the competition for generation of acyl-enzyme intermediate is needed to determine inhibition constants for the acetyl-oxa/aza(dethia)CoA analogs with FabH for the transthiolation reaction. However, such assays are inherently based on single-turnover kinetics and outside the scope of this report. Nevertheless, our data differ from fluoroacetyl-CoA hydrolase, where acetyl-aza(dethia)CoA is an inhibitor on the same order of magnitude as the malonyl-CoA Km. Furthermore, other examples where acetyl-aza(dethia)CoA has binding properties similar to acetyl-CoA are citrate synthase, where the acetyl-aza(dethia)CoA Ki is reported to be 1.6 µM, which is lower than the 8.5 µM acetyl-CoA Km, and phosphotransacetylase with a Ki of 130 µM and acetyl-CoA Km of 210 µM. Together these studies reveal the diverse behavior of acetyl-oxa/aza(dethia)CoA analogs with enzymes and their potential usefulness for enzyme structure-function studies.