Acivicin, an inhibitor of L-glutamine-dependent amidotransferases, is active against the murine L1210 and P388 leukemia models. Cytidine triphosphate synthetase has been proposed as the primary target for this agent. Our results demonstrate that Acivicin is also an inhibitor of de novo pyrimidine biosynthesis. This inhibition results in the depletion of pyrimidine deoxyribonucleoside triphosphate pools and explains the effect of this agent on DNA synthesis. Further, Acivicin is synergistic with N-(phosphonacetyl)-L-aspartic acid, another inhibitor of de novo pyrimidine synthesis. The combination of these agents results in a more than additive depletion of deoxycytidine triphosphate pools which may account for their synergism in inhibiting cellular growth. Thus, the inhibition of de novo pyrimidine synthesis by Acivicin may be useful in modulating the effects of certain antimetabolites or other inhibitors of this pathway.