The amino-terminal 8-kDa domain of DNA polymerase ␤ functions in binding single-stranded DNA (ssDNA), recognition of a 5-phosphate in gapped DNA structures, and as a 5-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure. Singlestranded DNA binding was probed by photochemical cross-linking to oligo(dT) 16 . Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5 terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys 35 is involved in 5-phosphate recognition of DNA polymerase ␤. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA.