Cytosine arabinoside (araC) is a potent antileukemic agent that is misincorporated into DNA in the course of its action. We have developed a chemical synthetic method that allows site-specific introduction of araC into synthetic DNA oligomers. We describe here the utilization of these oligomers as primer/template substrates for in vitro DNA synthesis reactions and as fragments for DNA ligation. These studies were undertaken to investigate the manner in which sites of araC misincorporation constitute sites of DNA dysfunction. AraCMP at the primer terminus dramatically reduced the rate of next nucleotide addition for Escherichia coli polymerase I (Klenow fragment) (Pol I), T4 polymerase, HeLa cell polymerase alpha 2 (Pol alpha 2), and AMV reverse transcriptase. Polymerases with associated 3'-5' exonuclease activity preferentially excised araCMP from the primer terminus prior to chain elongation. AraCMP-terminated fragments were ligated more slowly than control fragments by T4 DNA ligase. AraCMP located at an internucleotide site in the template markedly slowed replicative bypass for Pol I, T4 polymerase, and Pol alpha 2, but not for reverse transcriptase. Synthesis was partially arrested after insertion of the correct nucleotide opposite the lesion site. These results suggest a complex mechanism for the inhibition of DNA replication by araC when it is misincorporated into DNA.