A series of alkylating phosphoramidate analogs of 5-fluoro-2'-deoxyuridine has been prepared and their growth inhibitory activity evaluated against murine L1210 leukemia and B16 melanoma cells in vitro. These compounds were designed to undergo intracellular release of the phosphoramidate anions, which it was hoped would function as irreversible inhibitors of thymidylate synthase. The expectation was that binding of the nucleoside moiety would be followed by alkylation of the enzyme via the phosphoramidate. The chloride, bromide, iodide, and tosylate analogs were highly potent inhibitors of L1210 cell proliferation, with increased inhibition observed at both higher drug concentrations and longer exposure times. Addition of thymidine completely reversed the inhibition for all compounds, suggesting that these compounds are acting via inhibition of thymidylate synthase. Although the nonalkylating morpholine analog 1f was ca. 50-fold less potent than the methyl(chloroethyl)amino compound, the piperidine analog 1g was only 2-fold less potent, confirming that nitrogen basicity may be as important as the presence of an alkylating group. Addition of thymidine reversed the growth inhibition of the morpholine and piperidine analogs, suggesting that these compounds may also undergo intracellular conversion to 5-fluoro-2'-deoxyuridine 5'monophosphate. The thymidine and deoxyuridine derivatives 2 and 3 showed minimal growth inhibition in the L1210 assay. The alkylating analogs showed modest cytotoxicity against B16 melanoma cells, and the potency of the analogs was more dependent upon the alkylating moiety than on the 5-substituent.