Thymidine 5'-phosphate (TMP) derivatives with masked phosphate groups were synthesized in tritiated form from [methyl-3H]thymidine. They were of interest as models for 5' nucleotide derivatives that might be able to permeate mammalian cells and then liberate intracellular antimetabolite 5' nucleotides by loss of the masking groups. Mouse L fibroblasts were grown in vitro in the presence of 1 mM 5'-amino-5'-deoxythymidine, which was found to suppress greater than 99% of cellular thymidine kinase activity while inhibiting the rate of cell division by only 30%. The TMP derivatives were less effective than thymidine in labeling the deoxyribonucleic acid (DNA) of the L cells. The labeling was inhibited 95-99% by 5'-amino-5'-deoxythymidine, indicating that it represented incorporation into DNA of [3H]thymidine formed from degradation of the test compounds. No evidence was obtained that the compounds acted as sources of intracellular TMP by cell permeation followed by loss of phosphate blocking groups. Similar studies yielded no evidence that the bis(m-nitrophenyl) ester of TMP produced intracellular TMP by that route in the LM(TK-) strain of L cells that are genetically deficient in thymidine kinase.