Asparaginase II (L-asparagine amidohydrolase, EC 3.5.1.1) activity of cells from stationary phase cultures of Saccharomyces cerevisiae is very low. When these cells are inoculated into minimal medium, asparaginase II specific activity rises rapidly and reaches a maximum after 9-10 h. During the next 2.5-3 h, a rapid decrease in asparaginase II specific activity occurs. The enzyme does not appear to be secreted into the medium or to be reabsorbed into the cell. Addition of protease inhibitors at the time of maximum activity partially or totally prevents the loss of asparaginase II. L-1-Tosylamide-2-phenylethyl chloromethyl ketone decreases the rate of loss. The sulfhydryl reagents p-hydroxymercuribenzoate and iodoacetamide inhibit the loss of asparaginase II. However, addition of EDTA causes a further increase in activity. This increase is due to de novo protein synthesis. The effect of EDTA can be reversed by the addition of Zn2+. The most likely explanation for the rapid loss of asparaginase II is proteolytic degradation by a Zn2+-dependent, thiol protease or peptidase.