Four known polyacetylenes (NC-[C≡C]₂-CH=CH-CO₂H, H[C≡C]₃-CH=CH-CO₂H, HOCH₂-[C≡C]₂-CH=CH-CO₂H, and HO₂CCH=CH-[C≡C]₂-CH=CH-CO₂H) and four benzene derivatives (p-nitroanisole, p-nitrobenzaldehyde, 4-chloro-3-methoxybenzaldehyde, and 3-chloro-4-methoxybenzaldehyde) have been isolated from the culture fluids of the fungus Lepista diemii Singer. The aromatic compounds have not been previously reported as fungal metabolites. Analysis of the ether extract of the culture fluid revealed the presence of the four known polyacetylenic acids (I-IV; R=H), with two more present in trace amounts but not identified. Diatretyne 2 (I; R=H) and diatretyne 3 (III; R=H) were the main metabolites, and their concentrations in the culture fluid varied considerably with the conditions of fungal growth. The occurrence of diatretynes in Clitocybe and Lepista species has been tentatively proposed to support Singer's classification of the Agaricaceae family of fungi. The biogenesis of the L. diemii polyacetylenes is being investigated. In addition to the polyacetylenic acids, the fungus produced a variety of benzenoid metabolites in low concentrations, and four of these (V-VIII), not previously reported as fungal metabolites, were isolated from the neutral fraction of the extract. The production of these benzenoid metabolites also varied with growth conditions. p-Nitrobenzaldehyde could be a degradation product of chloramphenicol; however, chloramphenicol was not detected in the relevant fractions of the neutral part of the extract by comparative thin-layer chromatography (limit of detection 3 µg per liter of culture fluid). None of the metabolites were detected during the first 10 days of fungal growth.