Seven Mycobacterium strains were grown statically on salts-glycerol-asparagine (Sauton) or on salts-glucose-glutamate (Sym) media. At desired time of incubation, the bacteria were washed with water, disintegrated with powdered corundum and in resulting cell-free extracts L-asparaginase activity was determined by the Conway method. The majority of experiments were performed on M. phlei which exhibited considerable rise in L-asparaginase activity with increasing age of the culture. This change did not occur on Sym medium because of Zn2+, which proved to abolish the effect of the enzyme induction in vivo but did not inhibit the activity in vitro. Addition of rifampicin to Sauton culture media resulted in a low enzyme level. Exogenous asparagine and glycerol were not indispensable for the enzyme synthesis and could be replaced by glutamate and glucose, respectively.