The response surface method (RSM) was used to optimize the medium for the production of inulinase by Kluyveromyces sp. Y-85. The inulinase production was adequately approximated with a full quadratic equation obtained from a four-factor-five-level central composite design. Analyses of the quadratic surfaces showed that in a 24-h fermentation at 30 degrees C, the maximum inulinase activity 59.5 U/ml appeared at extract of Jerusalem artichoke, urea, beef extract, corn steep liquor concentrations 8.0%, 2.0%, 0.2% and 4.0%, respectively. We further investigated the fermentation in 15 l fermenter and scaling-up in 1000 l tower fermenter. The inulinase activity in the scaling-up was 68.9 U/ml, which was the highest production reported at present, indicating that Kluyveromyces sp. Y-85 was an excellent strain for industrial production.