The effect of the alkylating agent tris-(β-chlor- ethyl)-amine on glucose utilization in ascites tumor cells in vitro has been investigated. The influence of the unhydrolyzed and of the hydrolyzed agent was compared. Both affect the observed metabolic para- meters in comparable ways: They cause an increase in the rate of the oxidation of glucose C-6 to CO2, an increase in the level of glycolytic intermediates preceding the glyceraldehyde-phosphate dehydrogenase reaction, and an inhibition of lactic acid formation. In both cases the inhibition of lactic acid formation is counteracted by the addition of nicotinamide. Since the hydrolyzed drug, in contrast to the unhydrolyzed agent, does not exert a carcinostatic effect in vivo, the data provide additional evidence for the view that the cytostatic action is not the consequence of an inhibition of glycolysis