Mammalian DNA polymerase beta (beta-pol) is considered to be part of the DNA repair machinery. However, there have been conceptual problems with this idea because beta-pol does not completely fill some gapped substrates, and unlike other DNA polymerases, beta-pol adds just one dNMP for each cycle of binding to the DNA substrate, incorporation, and product release (distributive synthesis). To examine the questions of complete gap filling and gap recognition by beta-pol, we designed template-primer substrates with a range of gap sizes from 1 to 53 template residues between the 3'-OH primer and a downstream polynucleotide. M13mp18(+) single-stranded DNA was used as template, and synthetic deoxyoligonucleotides were used as primers and downstream polynucleotides. We find that beta-pol can completely fill gaps and that the gap-filling activity on substrates with gaps of up to 6 nucleotides is highly processive rather than distributive; processive synthesis to fill a gap strictly required 5'-phosphate on the 5'-moiety of the gap. Similar specificity for filling short gaps was not observed with other DNA polymerases. These results on substrate specificity and the mechanism of beta-pol suggest distinct requirements for the role of beta-pol in vivo.