Fungal metabolites were extracted from two isolates of Penicillium commune and two isolates of Aspergillus versicolor grown on yeast extract agar (YES) and Czapek yeast extract agar (CYA). Optimized conditions for analysing the extracts by high-performance capillary electrophoresis (HPCE) were obtained by experimental designs. The following nine factors were examined by a two-level fractional factorial design: concentration of the buffer ions PO 3− 4 and B 4O 2− 7, ionic strength of buffer, buffer pH, addition of sodium dodecyl sulphate (SDS) and sodium deoxycholate (SCD), addition of acetonitrile and methanol and voltage. Four factors significantly influenced the separation of peaks in all the fungal extracts. For optimization of the HPCE method a subsequent response-surface experiment with the important factors was made with an extract from an isolate of Aspergillus versicolor. Optimum separation conditions were obtained, which gave good resolution of the components in the extract.