Target and non-target response of mouse L-cells to aminofolates, aminopterin and amethopterin, as well as to a 5-methylquinazoline antifolate CB 3703 were compared. When added into the culture medium for 48 hr, each of those antifolates strongly inhibited the cell proliferation; the respective 1 0.5 values were of the order of 10 −8M for aminopterin and of the order of 10 −7 M for amethopterin and the quinazoline antifolate. However, even at concentrations which did not limit cell growth, each of the antifolates accumulated intracellularly far above the level of dihydrofolate reductase, and this accumulation was concentration dependent. Simultaneously, the activity of the target enzyme, dihydrofolate reductase, decreased considerably with a concomitant decrease in the activity of lactate dehydrogenase and in lactate content, whilst the content of pyruvate increased in the cells. Consequently, the ratio of lactate to pyruvate decreased. At growth-limiting concentrations, the antifolates did not only reduce the activity of dihydrofolate reductase nearly to zero, but also decreased to a smaller extent the activities of other folate-related enzymes such as methionine synthetase and serine hydroxymethyltransferase, and changed those of thymidylate synthetase and formyltetrahydrofolate synthetase to a greater or lesser degree and in a differentiated manner. Thus, the quinazoline antifolate substantially diminished the activity of thymidylate synthetase, whose activity was apparently enhanced by both aminofolates as compared with the respective controls. On the other hand, the activity of formyltetrahydrofolate synthetase was elevated by the quinazoline antifolate, being greatly decreased by aminopterin and only slightly lowered by amethopterin. Each of the antifolates also caused a significant decrease in the activity of glucose-6-phosphate dehydrogenase and a sharp decrease in that of lactate dehydrogenase. A simultaneous decrease in lactate content and an increase in pyruvate content led to a significant decrease in the ratio of lactate to pyruvate, indicating an increase in intracellular NAD + content and glycolysis impairment in the L-cells. In conclusion, we suggest considering the ratio of lactate to pyruvate as a kind of simple measure of the non-target effects of antifolates in mammalian cells.