Culture characteristics and metabolomic profiling (on the basis of gas chromatography-mass spectrometry) of 3 strains of Lignomyces vetlinianus were studied. Growth rate, macromorphology, and micromorphology of mycelia grown on various media are described. More than 60 compounds were detected in the mycelial extracts, including amino acids, organic acids that are active during the tricarboxylic acid cycle, sugars, fatty acids, sugar alcohols, and sugar acids. Principal component analysis of low-molecular-weight compounds in mycelial methanol extracts of L. vetlinianus strains at different stages of growth demonstrated that the pattern of mycelial metabolomes grouped by age of the culture indicates a significant relation between the development of the culture and the specificity of its metabolite spectrum. Slow-growing cultures develop gradually and are characterized by several changes in metabolite states. The pattern of points is grouped more tightly for fast-growing strains. The production of crystal-like aggregates was observed for aging mycelia at the stationary phase of growth. These aggregates were isolated from mycelia and identified as clusters of 4,6-dimethoxy-phthalide. The molecular structure of this substance was confirmed by nuclear magnetic resonance analysis. The results show that the concentration of 4,6-dimethoxy-phthalide increased during cultivation. Fruiting bodies contained very small amounts of 4,6-dimethoxy-phthalide compared with amounts in mycelia. It can be assumed that L. vetlinianus is a powerful natural producer of phthalides of biotech-nological interest and can be used as a model to study phenolic metabolism in fungi.