The purpose of these studies was to characterize the effect of the new fluoropyrimidine nucleoside 5'-deoxy-5-fluorouridine (5'dFUrd) on macromolecular processes in correlation with its cytotoxicity in Ehrlich ascites tumor cells. Following a 2-h exposure, 5'-dFUrd exhibited an LD50 (as determined by clonogenicity) of 48 microM. In cells supplemented with 10 microM dThd, the LD50 for 5'-dFUrd increased to 660 microM. DNA synthesis was markedly and rapidly suppressed by all cytotoxic concentrations of 5'-dFUrd. There was no apparent direct measurable effect of 5'-dFUrd on either RNA or protein synthesis, although both were suppressed 24 h after the drug exposure. Thymidylate synthetase activity was completely inhibited by all cytotoxic concentrations of 5'-dFUrd. FUra incorporation into RNA was also measured and appeared to correlate with the dThd-nonreversible toxicity of 5'-dFUrd. These studies indicate that the mechanism of 5'-dFUrd cytotoxicity is directly analogous to that reported for 5-fluorouracil. The inhibition of thymidylate synthetase leading to an inhibition of DNA synthesis was the most potent cytotoxic mechanism (i.e., dThd-reversible) for 5'-dFUrd, and was found to be highly time-dependent. Higher concentrations of 5'-dFUrd resulted in dThd-nonreversible toxicity, which appeared to be related to the incorporation of FUra into RNA.