Previous studies in this laboratory have utilized a chemically synthesized photolabile analog of benzoyl-coenzyme A, p-azidobenzoyl-coenzyme A (PAB-CoA), to label the regulatory sites of pyruvate carboxylase isolated from yeast (D. E. Myers, and M. F. Utter, 1978, Fed. Proc. 37,856). The following report describes a convenient enzymatic synthesis of PAB-CoA, catalyzed by a medium-chain fatty acid:CoA ligase enzyme from beef liver mitochondria. PAB-CoA synthesis is monitored by thin-layer chromatography; the purified product is subsequently isolated by conventional ion-exchange and gel-filtration techniques. Enzymatically synthesized PAB-CoA is identical to the chemically synthesized material as evidenced by thin-layer chromatography, spectral characteristics, and the ability to activate pyruvate carboxylase from yeast. The general enzymatic synthesis has been used to synthesize oand m -azidobenzoyl-CoA, as well as the related compound, p-azidophenylacetyl-CoA. All of these photolabile analogs of benzoyl-CoA activate pyruvate carboxylase from yeast and have potential use as modifiers of the regulatory sites of this enzyme as well as of other enzymes affected by CoASH. This enzymatic synthesis is also useful for preparing material of very high specific radioactivity.