Asparaginase II of Saccharomyces cerevisiae is a cell wall mannan containing glycoprotein. Recent studies have demonstrated that asparaginase II activity increases in exponentially growing cell cultures and then decreases as the cells enter the stationary phase. Enzyme inactivation has been attributed to a Zn2+-dependent protease which is synthesized de novo during the late exponential phase [Pauling, K. D., & Jones, G. E. (1980) J. Gen. Microbiol. 117, 423–430; Pauling, K. D., & Jones, G. E. (1980) Biochim. Biophys. Acta 616, 271–282]. We have investigated the mechanism of asparaginase II inactivation using both whole cell suspensions and highly purified enzyme. Our data indicate that the rate of asparaginase II inactivation in cell suspensions is primarily influenced by pH changes that occur as a consequence of cell growth and glucose fermentation and that enzyme inactivation is not dependent on Zn2+ or on de novo protein synthesis. Also, in vitro studies with purified enzyme show kinetics of inactivation that are similar to those observed in vivo. Consequently, involvement of a yeast protease in the inactivation process is relatively unlikely.