Primary cultures of rat hepatocytes were exposed to various concentrations of L-asparaginase derived from Escherichia Coli. Protein synthesis was inhibited by about 33% and cellular glutamine was reduced proportionally to the enzyme concentration. However, protein synthesis was inhibited only by amounts of enzyme able to reduce glutamine to critical levels below 10 nmol/mg cell protein. These data suggest that the glutaminase activity which probably contaminates E. coli asparaginase may be responsible for reduced liver protein synthesis.