9-beta-D-Arabinofuranosyladenine (ara-A) is an inhibitor of DNA replication with antitumor and antiviral activity. The molecular basis for this inhibitory effect has remained unclear. The present work has examined the effects of 9-beta-D-arabinofuranosyladenine-triphosphate on DNA polymerase-beta. These studies were performed on different M13 phage DNA templates. The findings demonstrate that 9-beta-D-arabinofuranosyladenine is incorporated into the elongating DNA strand by DNA polymerase-beta. The findings also demonstrate that the incorporated 9-beta-D-arabinofuranosyladenine residue acts as a relative chain terminator. Furthermore, the relative chain-terminating effects of this agent are sequence specific and reversed by competition with deoxyadenosine-triphosphate for incorporation into the DNA strand. These findings are in concert with hydrogen bonding differences of the incorporated arabinosyl moiety which alters reactivity of the chain terminus and thereby inhibits elongation. These findings are also in agreement with recent studies of 1-beta-D-arabinofuranosylcytosine and provide insights into the sequence specific effects of these agents.