A simple and rapid standardized micro-scale extraction procedure has been developed to prepare extracts from fungal cultures for high-performance liquid chromatographic (HPLC) analysis. The method is based on ultrasonic extraction of three 6-mm plugs cut from a culture using 0.5 ml of solvent followed by a simple solvent change, filtration and injection. Approximately 5 min of work is involved in the extraction and work-up process and the extract can prepared for HPLC analysis within 60-70 min. The method has been used for determination of chromatographic metabolite profiles from 395 fungal isolates, including all terverticillate Penicillium species, cultivated on both Czapek Yeast Autolysate agar and Yeast Extract Sucrose agar. The concentration of the extracts proved to be sufficient to determine all secondary metabolites reported to be produced by these species using HPLC with diode array detection. These findings were confirmed by analyses of 132 pure metabolite standards.