The naturally occurring nucleoside analogue arabinosyl thymidine is known as an anti-herpes and anti-cancer agent. The biologically active form is arabinosyl thymidine triphosphate (Ara-TTP), which inhibits cellular and viral DNA-polymerases and thus interferes with DNA replication. Using two murine erythroleukemia cell lines, Friend cell clone F4-6 and F4-12N, the latter being thymidine kinase deficient (TK-) cells transformed to a TK+ phenotype with the HSV TK gene, we have determined 1) the role of cellular and herpes simplex virus thymidine kinase (HSV TK) in the uptake of Ara-T into the cells; 2) the subsequent phosphorylation of intracellular Ara-T to Ara-TMP, Ara-TDP and Ara-TTP; 3) the incorporation of Ara-TTP into the DNA. Incorporation into DNA was studied under different conditions, including selective inhibition of the different cellular DNA polymerases by aphidicolin (that inhibits polymerases alpha and delta) and dideoxythymidine (that preferentially inhibits polymerases beta and gamma). The uptake of Ara-T into the methanol soluble pool of the cells depends upon its phosphorylation to Ara-TMP, which is more efficiently performed by the HSV TK than by the cellular TK, thus explaining the sensitivity of HSV infected cells to Ara-T. However, using increasing concentrations of Ara-T, we have shown that phosphorylation also occurs in normal control cells due to the cellular thymidine kinase. More than 90% of Ara-T is phosphorylated in the cell, and more than 60% of total Ara-T(MP, DP, TP) exists in the triphosphate form.(ABSTRACT TRUNCATED AT 250 WORDS)