Macrophomate synthase (MPS) catalyzes the reaction of oxaloacetate and 2-pyrones to give, over multiple steps, substituted benzoates. We detected a transient intermediate in the course of this transformation by monitoring the total time course either spectroscopically at 305 nm or by H-1 NMR. This species was trapped by quenching the reaction with acetonitrile and cooling the sample to 270 K; its structure was determined to be an allylic cyclohexadienol by a complete 2D NMR spectroscopic analysis. It is formed according to Michaelis-Menten kinetics at a rate that is as fast or faster than the decarboxylation of oxaloacetate, the first step in the reaction sequence. Dehydration of this compound to give macrophomate, which limits the rate of the overall process, is not catalyzed by MPS. Although these results clarify the final stages of MPS catalysis, they shed no light on the early C-C bond-forming step(s). As a consequence, other methods will be needed to resolve the controversy as to whether this enzyme functions as a natural Diels-Alderase.