Deazauridine inhibited growth of tumor cells in culture and in culture and in vivo; this agent was significantly more effective against L1210/AraC than against the parent sensitive line. Inhibition of growth of tumor cells in culture was prevented by uridine and cytidine and was partially alleviated by deoxycytidine, but not by deoxyuridine or thymidine. DeazaUR inhibited nucleic acid synthesis but not protein synthesis in tumor cells in culture; deoxycytidine alleviated inhibition of nucleic acid synthesis. The labeling of pyrimidine ribonucleotides by 6-14C-orotic acid was inhbited by deazaUR. DeazaUR treatment of tumor cells in culture resulted in increased uptake of cytidine-3H into RNA, whereas uridine-3H uptake into RNA was inhibited. Labelling of DNA by uridine-3H/ and cytidine-H was inhibited by deazaUR. Pools of CMP, CDP, and CTP decreased markedly during deazaUR treatment of L1210 cells in culture and in vivo. These observations in growing cells pointed to deazaUR inhibition of the synthesis of cytidylic acid. Deazauridine 5'-triphosphate was found to be an inhibitor of the synthesis of CTP from UTP catalyzed by enzyme preparations from L1210 cells. This observation is in agreement with those of McPartland et al.19 that deazaUTP inhibited CTP synthetase purified from calf liver. Deazauridine treatment of L1210 cells in culture stimulated the uptake of deoxycytidine-3H into DNA while inhibiting the uptake of 3H-labeled deoxyuridine, thymidine, deoxyadenosine, and deoxyguanosine. Intracellular pools of dCTP were decreased by deazauridine treatment in L1210 cells in culture and in vivo. Deazauridine 5'-diphosphate inhibited the enzymatic reduction of pyrimidine ribonucleoside 5'-diphosphates to the corresponding deoxyribonucleotides. These results are consistent with the view that deazauridine, after its uptake and intracellular phosphorylation, strongly inhibits the formation of CTP. This is considered to be the primary metabolic effect of the analog. A secondary effect appears to be an inhibition of dCTP formation.