A number of 5-substituted pyrimidine deoxyribonucleoside (dThd or dCyd) derivatives have been evaluated for their effects on the incorporation of dThd and dCyd into the nucleotide pool and nucleic acids of murine leukemia L1210 cells. Several observations indicate that the dThd kinase and dCyd kinase activity of the cells and the differential affinities of these enzymes for the pyrimidine deoxyribonucleosides determine the incorporation of dThd and dCyd into the cells: (i) dThd and dCyd were not incorporated into mutant L1210 cells deficient in either dThd kinase or dCyd kinase activity; (ii) for a series of 5-substituted dThd and dCyd analogues a strong correlation was found between their inhibitory effects on the incorporation of dThd or dCyd into cell material and their Ki/Km for dThd kinase and dCyd kinase (r = 0.92 and 0.97, respectively); (iii) inhibitors of DNA synthesis (i.e. araC) and RNA synthesis (i.e. actinomycin D) suppressed the incorporation of dThd, most likely due to an inhibitory activity at the dThd kinase level (through the allosteric action of dTTP or slow regeneration of dThd kinase).