A suitable strain and important factors influencing citric acid formation in yeasts were identified. Candida oleophila ATCC 20177 was chosen as the best citric acid producer from several Candida strains. Yields of 50 g/l citric acid were produced in shake flask and 80 g/l in fed-batch fermentations with 1.5 and 3 g/l NH4Cl under non-optimized conditions. Ammonium nitrogen was identified as the limiting substrate for citrate formation. Citric acid excretion begins a few hours after exhaustion of nitrogen in the medium. The importance of intracellular nitrogen limitation was clarified by elemental analysis of C. oleophila biomass. The nitrogen content of C. oleophila biomass decreased from 7.45% during the growth phase to 3.96% in the production phase. The biomass contained less carbon and more trace elements in the growth phase compared with the production phase. Relatively high intracellular NH4+ concentration of about 1.2 mg/g biomass (similar to37.4 mM) was found during the production phase. The low intracellular nitrogen content and increase of intracellular NH4+ concentration, possibly caused by proteolysis following extracellular nitrogen exhaustion, trigger citric acid production. Intracellular nitrogen limitation and the increase in intracellular NH4+ concentration are the most important factors influencing citric acid formation in yeasts.