The production of acetylenic phenol metabolites in vitro by three strains of the ascomycete Eutypa lata, the causative agent of dying-arm disease in grapevines, has been investigated. Metabolite composition and yields differed significantly between strains and with growth medium but usually reached a maximum after 24-30 days of fungal growth. A general method for the analysis and identification of metabolites by gas chromatography/mass spectrometry of their trimethylsilyl ether derivatives was developed and individual compounds were quantitated by analytical high-performance liquid chromatography (HPLC) and separated by preparative HPLC. The phenolic aldehyde, eutypine (1), reported to be the grape phytotoxin, occurred in only one of the strains examined whereas the primary metabolite was the corresponding alcohol, eutypinol (2), the presumptive detoxification product. A novel metabolite was isolated as a major constituent, together with a minor component, and their structures were established by spectroscopic methods as a methoxyquinol, named eulatinol (4), and a chromene analog (9) of 2, respectively. The evidence suggests that 1 is not solely responsible for phytotoxicity in grapevines but that dying-arm disease may result from a suite of compounds elaborated by the fungus, with the composition dependent on fungal strain and nutritional source.