The xylanase gene from the rumen fungus Neocallimastix frontalis was expressed in Pichia pastoris and Pichia methanolica. Using a complex medium with medium replacement before induction and the maintenance of the methanol induction level at 0.5%, P. pastoris was able to produce about 5400 U/ml of xylanase after 10 days of induction. With P. methanolica, on the other hand, about 6200 U/ml of xylanase was reached after 10 days of induction using synthetic medium as first culture medium and then direct induction by continuous methanol feed at 1.8 ml l(-1) h(-1). In general, the advantages of using P. methanolica to produce the xylanase included higher protein production, the lack of medium replacement, and an ease of scale up. However, because the P. pastoris culture supernatant contained fewer secreted non-target proteins compared to P. methanolica, xylanase purification would be easier with the P. pastoris system. In addition, experiments involving methanol pulses suggested that a relationship exists among base feeding, methanol consumption and xylanase activity. (c) 2008 Elsevier Inc. All rights reserved.