An activity dependent on an intact bacteriophage T4 gene 61 is required, along with the T4 gene 41 protein, for the synthesis of ribonucleotide primers in an in vitro T4 DNA replication system. In this paper, we present a method for purification of the protein catalyzing this gene 61-dependent activity based on an assay for primer-dependent DNA synthesis by the T4 DNA replication proteins. The T4 gene 32 helix-destabilizing protein influences the chromatographic behavior of 61 protein. The purification of 61 protein to near homogeneity by the scheme presented requires the presence of 32 protein in crude extracts. The isolated 61 protein is basic, with a molecular weight of 44,000, and is active as a monomer. Ribonucleotide primer synthesis shows a linear dependence on 61 protein concentration, but a sigmoidal dependence on 41 protein concentration. The dependence on 41 protein concentration is linear, however, if the 41 protein is first "activated" by incubation at high concentration in the presence of rGTP. Using this "activated" 41 protein and purified 61 protein, we show a stoichiometric relationship between the two proteins in the priming reaction consistent with the existence of a priming complex comprising an oligomer of 41 protein and a 61 protein monomer.