Plasmodium falciparum merozoite surface protein 3 (MSP3) is a leading blood-stage malaria vaccine candidate. Vaccination with Pichia pastoris derived recombinant MSP3 protected Aotus nacymai monkeys from the parasite's lethal challenge and the post-challenge antibody titer against MSP3 correlated with protection. In our preliminary attempts to produce this vaccine in fermentors, little or no expression of MSP3 was observed in chemically defined media, although the same P. pastoris strain produced MSP3 in complex media. Our goal is to develop a Phase I/II clinical manufacturing process in completely chemically defined media because of the concern of potential prion contamination in complex media containing animal derived products. Here, we report our investigations into various factors to improve the yield of MSP3 in defined media. We found that an induction pH (pH(i)) 6.8 yielded MSP3 at 434 mg/L whereas there was no product at pH(i) <= 5, though cell growth was the same in all pH(i) levels examined. High levels of NH4+ consumed at pH(i) 6.8 were directly correlated to the enhanced MSP3 production. Furthermore, an additional 3.5-fold increase in the yield of MSP3 was obtained by addition of casamino acids at pH(i) 6.8. No direct correlation was observed between protease activity in the culture supernatants and lack of MSP3 expression. Neither high P. pastoris biomass generated at a high specific growth rate (0.04/h) nor low induction temperatures during induction improved yield. Nitrogen source was the most important factor affecting expression of MSP3 in defined media. Published 2005 Wiley Periodicals, Inc.