The biosynthetic introduction of oxygen in position 7 of brefeldin A, a structurally unique macrolide with an alicyclic ring, was studied. [4-2H]Brefeldin C was prepared efficiently from brefeldin A. A high incorporation ratio of the labeled brefeldin C into brefeldin A by Eupenicillium brefeldianum clearly indicates that the oxygen in position 7 of brefeldin A does not contribute to the cyclopentane ring formation but is introduced during the last step of brefeldin A biosynthesis.