Human cystatin C is a cysteine-proteinase inhibitor with potential as a therapeutic protein for treating viral and bacterial infections, cancer, some vascular diseases and rheumatoid arthritis. We selected a Mut s strain of Pichia pastoris to express human cystatin C under regulation of the methanol-inducible AOX1 gene promotor. The effects of pH and of feeding both methanol and glycerol during induction were investigated in a 2-liter bioreactor. A concentration of 54 μmoles l - 1 , equivalent to 0.72 g l - 1 of active human cystatin C, was achieved at pH 6.0 while feeding 1.8 g l - 1 h - 1 methanol as the sole carbon source during induction for 96 h. Cystatin C productivity was increased from 0.60 μmole l - 1 h - 1 for methanol-only feed to 0.96 μmole l - 1 h - 1 when 2.1 g l - 1 h - 1 of glycerol was fed together with 1.8 g l - 1 h - 1 methanol during induction. Glycerol feeding increased cystatin C concentrations early on in the induction phase, but cystatin C concentrations leveled off and then decreased with induction time. When 2.1 g l - 1 h - 1 of glycerol was fed together with 1.8 g l - 1 h - 1 methanol a maximum concentration of 45 μmoles l - 1 (0.65 g l - 1 ) of cystatin C was produced. Thus, a Pichia pastoris fermentation system for high-level expression of human cystatin C has been developed. The highest concentrations were obtained on methanol feeding only, but adding glycerol during the induction phase increased volumetric productivity.