Pools of uridine triphosphate and cytidine triphosphate are greatly (90%) reduced in cultured L1210 cells exposed to N-(phosphonacetyl)-L-aspartate (PALA) or pyrazofurin; the concentration of the deoxynucleotides deoxycytidine triphosphate, deoxythymidine triphosphate, and deoxyguanosine triphosphate also decreases, but deoxyadenosine triphosphate pools are enlarged. Associated with these pool depletions is a pronounced inhibition of DNA synthesis even when pools are only moderately reduced; RNA synthesis is only slightly inhibited under these same conditions. DNA synthesis in permeabilized preparations of L1210 cells was also more sensitive than was RNA synthesis when the concentrations of ribonucleotide and deoxyribonucleotide triphosphates presented were equivalent to those found in PALA- or pyrazofurin-treated cells. The specific sensitivity to depletion of DNA precursors was also seen in protection of both DNA synthesis and growth of L1210 cells by deoxycytidine and thymidine. This supplement restored deoxycytidine triphosphate, deoxythymidine triphosphate, and deoxyguanosine triphosphate pools to normal but of course did not affect the marked depletions of uridine triphosphate and cytidine triphosphate or the less marked effect of PALA on RNA synthesis. The relative ability of PALA to reduce uridine triphosphate and cytidine triphosphate pool size in L1210 ascites and Lewis lung carcinoma in vivo correlates with the intrinsic sensitivity to this agent.