Substrate specificity in the novel biotransformation from 2-pyrone derivatives to substituted benzoic acids by Macrophoma commelinae (IFO 9570) was investigated by means of feeding experiments with various compounds. Among them, 2-pyrone derivatives substituted by an electron-donating group at C-4, by an electron-withdrawing group at C-5 and by an alkyl group at C-6 were converted to the corresponding benzoic acid derivatives in fairly good yields. The C₃-unit precursors and intermediates were examined in stationary or shaking culture. Based on the experimental results obtained, a mechanism for this unique reaction is proposed.