The T4 bacteriophage gene 41 protein is known from genetic analysis to be essential for phage DNA replication in vivo. It became possible to monitor the activity of this protein during purification after development of an "in vitro complementation assay," which measures its stimulation of DNA synthesis in a concentrated crude lysate prepared from Escherichia coli cells infected with a T4 bacteriophage mutant in gene 41 (L. Moran and B. Alberts, manuscript in preparation). In this report, a purification procedure involving three chromatographic steps is described which reproducibly yields a 90% homogeneous preparation of this rather unstable protein. The major polypeptide chain present (58,000 daltons) is shown to cosediment with a DNA-dependent GTPase (and ATPase) activity, and to induce extensive in vitro DNA synthesis on both single- and double-stranded DNA templates when incubated with our preparations of five other purified T4 DNA replication proteins (plus deoxyribonucleoside and ribonucleoside triphosphates).