The lipases of the Rhizopus species family are important and versatile enzymes that are mainly used in fat and oil modification due to their strong 1,3-regiospecificity. Inexpensive synthetic medium was used for the production of Rhizopus oryzae lipase in the methylotrophic yeast Pichia pastoris. Methanol accumulation inside the bioreactor has previously been shown to negatively influence the production level. Three different methanol fed-batch strategies for maintaining the methanol concentration within optimal limits have been assayed in high-density cultures. One methanol feeding strategy, which is based on the monitoring of the methanol concentration by gas chromatography, resulted in a 2.5-fold higher productivity compared to an initial cultivation, where the feeding rate was adjusted according to the dissolved oxygen concentration (DO) in the supernatant. Finally, productivity could be further increased by introducing a transition phase that involved the simultaneous feeding of glycerol and methanol followed by a single methanol feed. This optimized strategy resulted in the highest productivity (12888 U l(-1) h(-1)), which is 13.6-fold higher than the DO-based strategy.