A DNA-unwinding protein has been purified to homogeneity from E. coli. This protein has a molecular weight of about 22,000, as judged by its electrophoretic mobility on polyacrylamide gels containing sodium dodecylsulfate, and it appears to be present in about 800 copies per log-phase cell. It binds tightly and cooperatively to single-stranded DNA, and much less tightly, if at all, to RNA or double-stranded DNA. Like the T4 gene-32 protein characterized previously, the E. coli DNA-unwinding protein depresses the melting temperature of double-stranded DNAs, with regions rich in A-T base-pairs being preferentially melted. The E. coli protein strongly stimulates in vitro DNA synthesis by E. coli DNA polymerase II on appropriate templates; however, no stimulation is found with purified polymerases I or III of E. coli, or with T4 DNA polymerase. In contrast, gene-32 protein stimulates only the T4 DNA polymerase in a parallel assay.