Although the quality of nitrogen source affects fermentation product formation, it has been managed empirically, to a large extent, in industrial scale. Laboratory-scale experiments successfully use the high-cost proline as a nonrepressive source. We evaluated urea as a substitute for proline in Saccharomyces cerevisiae ure2dal80 fermentations for asparaginase II production as a model system for nitrogen-regulated external enzymes. Maximum asparaginase II levels of 265 IU/L were observed in early stationary-phase cells grown on either proline or urea, whereas in ammonium cells, the maximum enzyme level was 157 IU/L. In all cases, enzyme stability was higher in buffered cultures with an initial pH of 6.5.