AbstractBy feeding growing cultures of Penicillium brefeldianum Dodge with [‐14C]‐acetate radioactive brefeldin A (C16H24O4) was obtained. By extensive degradation of [14C]‐brefeldin A, the individual radioactivity of 12 of the 16 carbon atoms was determined quantitatively. The pattern of the distribution of the radioactivity shows that 8 acetate units have been incorporated into the metabolite in a regular manner. These findings suggest an octaketide as an intermediate. Possible mechanisms for the formation of the trans‐fused cyclopentane ring are discussed.