PURPOSE: The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has proven to be one of the most effective agents available for the treatment of acute leukemia although the precise mechanism by which ara-C induces cytotoxicity remains unclear. Our laboratory has previously isolated from human cells a DNA replication complex, termed the DNA synthesome, which is fully competent to orchestrate, in vitro, all of the reactions required to efficiently and faithfully replicate DNA. Using this system and the active metabolite of ara-C, ara-CTP, we demonstrated that the human DNA synthesome can efficiently incorporate ara-CTP into internucleotide positions of newly replicated DNA in vitro mimicking results obtained using intact cells and isolated nuclei. We then hypothesized that DNA polymerase auxiliary proteins, present within the DNA synthesome, may aid in incorporating this nucleotide analog into DNA. METHODS: To test this hypothesis, we utilized three distinct multiprotein complexes each of which contained human DNA polymerase alpha and examined with standard in vitro polymerase assays the effectiveness of ara-C in inhibiting various aspects of their polymerase function. RESULTS AND CONCLUSION: These polymerase-mediated elongation assays, which included ara-CTP- or ara-C-containing primers in the reaction mixture, showed that the rate of DNA elongation in the presence of ara-CTP was significantly enhanced when the DNA polymerase was associated with its auxiliary proteins, and that the elongation resulted in the formation of internucleotide ara-CMP. Nevertheless, the enhanced activities resulting from the association of these auxiliary proteins with polymerase alpha did not fully account for the remarkable efficiency with which the DNA synthesome incorporated ara-C into internucleotide positions during DNA replication.