An efficient fermentation medium producing a colourless product with high yields has been developed for the production of recombinant hirudin from Saccharomyces cerevisiae. Response surface methodology was applied to optimize the medium constituents. A 2(5-1) fractional factorial central composite design has been chosen to explain the combined effect of the six medium constituents, viz. yeast extract, peptone, casamino acids, ammonium sulphate, potassium phosphate and galactose, and to design a minimum number of experiments. The optimized medium produced 65.3 mg/l of r-hirudin in shake flask culture, which is 35% higher than the unoptimized medium. The colour of the purified product was influenced by the colour intensity of culture supernatant, which in turn depended upon the concentration of complex substrates used in the medium. The concentration of yeast extract has been reduced to 16 g/l in the optimized medium than other reported media containing 40 g/l yeast extract. The colour intensity of culture supernatant obtained by the optimized medium was reduced by 60% from the reported media containing 40 g/l yeast extract. (C) 2000 Elsevier Science Ltd. All rights reserved.