A protein that stimulates the enzymatic replication of duplex DNAs of recombinant phages and plasmids bearing the Escherichia coli origin of replication (oriC) has been isolated from an extract of E. coli. The isolated protein and the well-known protein HU, a histone-like DNA-binding protein, have identical polypeptide molecular weights and saturate the oriC replication assay at less than 40 dimers per template DNA circle. This level is one-tenth that needed to coat the template. Protein HU from the blue-green alga Anabaena is similarly active. Antibody specific for protein HU from E. coli inhibits replication promoted both by the reconstituted system and by a crude enzyme extract; in both assays, activity is restored by excess of the isolated protein. Cells lysed in 1 M KCl yield 32,000 dimers of the protein per cell, a number consistent with the reported abundance of HU. These data establish the identity of the isolated factor and protein HU and provide an indication of a function for HU in replication.