The bacteriophage T4 gene 61 protein is required, together with the gene 41 protein and single-stranded DNA, for the synthesis of the pentaribonucleotides that are used as primers for the start of each new Okazaki DNA fragment during T4 DNA replication. Using this priming activity as an assay, we have purified the 61 protein to essential homogeneity in milligram amounts. The priming activity was identified with the product of T4 gene 61 by using two-dimensional polyacrylamide gel electrophoresis to compare all of the T4-induced proteins in wild-type and mutant infections; the purified protein co-migrates with the only detectable protein missing in a 61- mutant infection. The purified 61 protein is shown to bind to the T4 helix-destabilizing protein (gene 32 protein) and to both single-stranded and double-stranded DNA. We have failed to detect any ribonucleotide polymerizing activity in either the 61 protein or the 41 protein alone; both the 61 and 41 proteins must be present to observe any synthesis of oligoribonucleotides.