Medium improvement for quereetinase production by Penicillium olsonii was investigated using a fractional factorial design in two sets of eight experiments with seven variable components each. The tested components were: rutin concentration, nitrogen source and its concentration, KH2PO4, NaCl, MgS04 and yeast extract concentrations, inoculum size, copper and metal salts concentrations and pH value. A final optimisation step, varying only yeast extract concentration, allowed six-fold improvement of the quercetinase activity with respect to initial medium, reaching a maximum activity of 0.708 mumol/min/ml after 9 days of cultivation. (C) 2004 Published by Elsevier Ltd.