The main biochemical determinants involved in cytosine arabinoside (Ara-C) metabolism were studied in one lymphoblastic (Reh) and two myeloid (HL60 and K562) human leukemic cell lines exhibiting various sensitivities to Ara-C, Reh being the most and HL60 the least sensitive. The level of intracellular Ara-C accumulation and Ara-CTP formation was far more important in Reh cells than in myeloid cell lines but was not closely related to deoxycytidine kinase activity or to deoxycytidine triphosphate pool size. The level of Ara-C incorporated into DNA was similar in the three cell lines. Ara-CTP formation correlated better with the cytotoxicity to clonogenic cells than did Ara-C incorporation into DNA. DNA polymerase alpha was moderately inhibited to various degrees, depending on the cell line; this moderate inhibition does not seem sufficient to explain the inhibition of DNA synthesis. The activity of DNA ligase, the enzyme joining the Okazaki fragments, which was not detected in Reh cells, was strongly inhibited by Ara-C in HL60 and to a lesser degree, in K562 cells. The inhibition of DNA ligase probably also contributes to the inhibition of DNA synthesis and, thus, to the cytotoxic effect of Ara-C and may explain the smaller size of DNA fragments observed following Ara-C treatment. The variations in each critical determinant observed in these three cell lines increase the complexity and plurality of the mechanisms of Ara-C action.