A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.