Analogues of 5'-deoxy-5-fluorouridine (DFUR, 1) were synthesized with the aim of evaluating the effect of structural modification of the 5' region of the ribofuranose moiety on the activity of these potential prodrug derivatives of 5-fluorouracil (FU). Reaction of the 2',3'-O-isopropylidene-5'-O-p-toluenesulfonyl derivative 9 of 5-fluorouridine (FUR, 2) with lithium dimethylcuprate and acidolysis of the isopropylidene group with 90% trifluoroacetic acid gave the ribohexofuranoside 3. Azide displacement of the tosylate group from 9, followed by reduction with triphenylphosphine and acidolysis with trifluoroacetic acid, led to the 5'-amino compound 4. Use of n-butanethiol as the nucleophile provided the thioether 5. Glycosylation of O2,O6-bis(trimethylsilyl)-5-fluorouracil with D-erythrose triacetate and stannic chloride in acetonitrile, followed by deblocking with NaOMe, yielded the erythrofuranoside 8, which may be viewed as a hybrid of 1 and the FU prodrug Ftorafur. Air oxidation of the 5'-OH group in 2 in the presence of platinum catalyst afforded the 5'-uronic acid 6. Compounds 3,6, and 8 were active against P-388 leukemia in mice, giving median increases in life span (ILS) of +63, +63, and +75% at doses of 600, 300, and 640 mg/kg (ip, qd × 4). On the same schedule against this tumor, FU gave a +73% ILS at 15 mg/kg and DFUR gave a +54% ILS at 320 mg/kg. As expected, FUR was much more potent than any of the other nucleosides, with a +75% ILS at the optimal nontoxic dose of 4 mg/kg. The cytotoxicity of the 5'-modified DFUR analogues toward L1210 mouse leukemia cells in culture was much lower than that of FU, the difference in ID50 values being as great as 1000-fold in the case of the 5'-uronic acid 6. The activity of these compounds in vivo, coupled with their low activity in vitro, was consistent with a prodrug mechanism and suggested that further studies on the effect of 5'-modification may be of interest as part of a broader search for improved prodrug derivatives of FU.