The pattern of incorporation of ({ }^{13} \mathrm{C}) from ({ }^{13} \mathrm{C})-labeled acetate into stipitatic acid by Talaromyces stipitatus was studied by ({ }^{13} \mathrm{C}) NMR. The results obtained were consistent with the biosynthetic routes previously elucidated for similar fungal tropolones. These studies, which both required and enabled the first complete ({ }^{13} \mathrm{C}) NMR assignments of stipitatic acid, also demonstrated that the decarboxylation step in the biosynthesis of stipitatic acid occurs after the formation of the tropolone ring. The biosynthetic pathway to stipitatic acid includes an oxidative expansion of the benzene ring of 3 -methylorsellinic acid to a seven-membered tropolone ring. As a prelude to mechanism-based enzyme inactivation studies, the mechanism of this step was reinvestigated by the biosynthetic incorporation of ({ }^{18} \mathrm{O}) into stipitatic acid from ({ }^{18} \mathrm{O}_{2}). The ({ }^{13} \mathrm{C}) NMR spectrum of the resulting (\left[{ }^{18} \mathrm{O}\right]) stipitatic acid showed an ({ }^{18} \mathrm{O})-induced isotope shift for the tropolone-ring carbonyl resonance with no evidence of ({ }^{18} \mathrm{O}) enrichment at the C-6 hydroxyl group. This result provides conclusive evidence in support of the earlier report that the ring expansion involves oxidation by a monooxygenase to form a hydroxymethyl intermediate, which undergoes a pinacol rearrangement to form the tropolone ring. 1995 Academic Press. Inc.