Torulopsis glabrata is a strain of yeast that is used for the industrial production of pyruvate. Determination of the optimal nutrient environment is vital for obtaining the most efficient production system. In this study, the fermentation parameters, gene transcription levels, activities of key enzymes and metabolites levels were analyzed when either urea or ammonium chloride was used as the sole source of nitrogen. Urea caused an increase in the dry cell weight (18%) and pyruvate productivity was significantly increased (14%). The transcription levels of CAGL0M05533g (DUR1,2), CAGL0J07612g (ZWF1), and CAGL0I02200g (SOL3) were upregulated, but CAGL0G05698g (GDH2) and CAGL0L01089g (GLT1) were down-regulated. The activities of urea amidolyase, NADPH dependent glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were increased by 380, 430, and 140%, respectively. The activities of arginase and glutamate synthase were decreased by 40 and 35%, respectively. The NADPH content was increased by 33%, whilst ATP content was decreased by 37%. This changed the intracellular levels of organic acids and amino acids. The results expand the understanding of the physiological characteristics of yeast species grown with different sources of nitrogen. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:19-27, 2014