Eucalyptus hemicellulose was hydrolyzed by treating eucalyptus wood chips with sulfuric acid. The hydrolyzate was used as the substrate to produce single-cell protein by growing Paecilomyces variotii IOC-3764 for 72 or 96 h. The influences of rice bran, ammonium sulfate and fermentation time were verified by a 2(3) full-factorial central composite design. At the optimum process conditions, the cell concentration was 12.06 g/l, which was obtained when the microorganisms were cultivated for 89, h in a medium composed of 10 g/l rice bran, 2.0 g/l nitrogen and 1.1 g/l sodium phosphate. The mathematical model Y = 10.65 + 2.40X(2) + 2.36X(3) + 1.16X(2)X(3) -2.10X(2)(2) - 1.06X(3)(2) describes biomass production by P. variotii in eucalyptus hemicellulosic hydrolyzate with a determination coefficient of R-2 = 0.9561, where X-2 and X-3 are ammonium sulfate and fermentation time, respectively.