A reversed-phase high-performance liquid chromatographic determination of profiles of mycotoxins and other fungal secondary metabolites has been developed. Penicillium, Aspergillus and Fusarium polyketides, terpenes and alkaloids have been emphasized. In a gradient elution, using water-acetonitrile containing 0.05% trifluoroacetic acid, 134 secondary metabolites were eluted evenly with retention times from 1.08 to 34.48 min. Metabolites with the same retention time were usually not produced by the same species. As UV detection at 254 nm was used, some mycotoxins (type A trichothecenes, viridicatumtoxin, peptide-like compounds and xanthomegnin) could not be detected. The method appears to be valuable for chemotaxonomic studies of fungi. Unpurified concentrated chloroform-methanol extracts of petri dish cultures analysed by the proposed method presented gave species-specific characteristic profiles of known and unknown secondary metabolites and mycotoxins.