Improved porcine interferon-alpha (pIFN-alpha) production by recombinant Pichia pastoris was achieved by culture conditions optimization in a 5-l bioreactor. The results indicated that the pIFN-alpha concentration, specific methanol consumption rate, specific activities of alcohol oxidase, formaldehyde dehydrogenase, and formate dehydrogenase could be significantly enhanced by decreasing induction temperature. The highest pIFN-alpha concentration (1.35 g l(-1)) was obtained by simultaneously controlling methanol concentration at 5 g l(-1) and induction temperature at 20 A degrees C, which was about 1.6-fold higher than the maximum obtained with previous optimal methanol concentration level (about 10 g l(-1)) when inducing at 30 A degrees C. The potential mechanisms behind low temperature and low methanol concentration effect on pIFN-alpha production may be ascribed to higher cell metabolic activity, more carbon flux towards pIFN-alpha production, and less intracellular/extracellular protease release.