The mitochondrial enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein (trifunctional protein) plays a major role in mitochondrial fatty acid oxidation. The enzyme complex consists of four molecules of alpha-subunit containing both hydratase and dehydrogenase domains and four molecules of beta-subunit containing the thiolase domain. The primary structure of a gastrin-binding protein (GBP) was highly homologous to that of the alpha-subunit of the trifunctional protein. Here, we report that gastrin inhibits the hydratase, dehydrogenase, and thiolase activities of the trifunctional protein. The gastrin/cholecystokinin receptor antagonist benzotript, which inhibited binding of gastrin to the GBP, also inhibited all three activities of the trifunctional protein. In addition, benzotript inhibits the activities of multifunctional enzymes having similar structures, such as the peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein and the Pseudomonas fragi fatty acid oxidation enzyme complex. This reagent, however, hardly inhibited various monofunctional enzymes involved in fatty acid oxidation.