Background: Pichia pastoris is a highly successful system for heterologous expression. During the induction stage, the ammonium ion released into the fermentation broth has a deep impact on cell growth and protein expression. The impact of NH4+ concentration on the expression of the Rhizopus oryzae lipase proAROL in P. pastoris was investigated. Results: The lipase activity under the optimum NH4+ concentration of 440 mmol/L reached 12,019 U/mL. Increased concentrations of NH4+ in the broth prevented the protease production, resulting in higher specific lipase activity in the supernatant. Furthermore, analysis of carbon metabolism and energy regeneration pattern revealed that under the definite NH4+. concentrations more carbon source (methanol) was consumed with surged AOX activity and then the higher energy and amino acid precursors demand for recombinant protein synthesis is compensated for by the TCA cycle. Conclusions: In this study, the R. oryzae lipase activity reaches the highest level ever reported under optimized NH4+ concentration and the analysis of the carbon metabolism provides useful information for future optimization of protein production by P. pastoris in a molecular level. (C) 2013 Elsevier Ltd. All rights reserved.