Three new metabolites, named macommelinal, macrophin and macrophic acid, were isolated from the culture broth of Macrophoma commelinae IFO 9570. Their structures were determined to be 2-(4-methoxy-6-methyl-2-oxo-2H-pyran-5-yl)ethanal, 3-methyl-2-butenoic acid (E)-[5-hydroxymethyl-4-methoxy-6-(2-methoxycarbonylethenyl)-2-oxo-2H-pyran-3-ly]methyl ester and 3-(4-methoxy-3, 5-dimethyl-2-oxo-2H-pyran-6-yl)-2-propenoic acid, respectively. 4 Acetyl-3-methoxy-5-methylbenzoic acid, now named macrophomic acid, was also isolated from a large-scale culture.The biosynthesis of the macommelin group, novel 5-substituted 2-pyrone metabolites, has been investigated by feeding experiments with [1-¹³C]-, [1, 2-¹³C₂]- and [1-¹³C, 2-²H₃]acetate and [2-¹³C]malonate. It was concluded that all these metabolites originated from a single straight tetraketide chain. Furthermore, the biogenetic mutual relationships of these metabolites including potential intermediates were estabished through incorporation experiments with ¹⁴C-labeled compounds.