A procedure has been developed which allows the T4 bacteriophage proteins corresponding to the products of genes 43, 44, 45, and 62 to be purified to near homogeneity from a single T4-infected cell lysate (greater than 90% single species as judged by sodium dodecyl sulfate polyacrylamide elctrophoresis). In these preparations, the major problem of removing all contaminating nucleases has been overcome. Each of the above proteins is known from genetic analysis to be essential for phage DNA replication. The protein product of gene 43 is T4 DNA polymerase, and its recovery can be monitored using a standard DNA polymerase assay. The other three gene products have been designated as "polymerase accessory proteins," since they directly enhance polymerase function on both single- and double-stranded DNA templates. Their activities were monitored by an "in vitro complementation assay," which measures the stimulation of DNA synthesis observed in a concentrated lysate of T4 mutant-infected Escherichia coli cells when the missing T4 wild type protein is added. Starting from 300 g of infected cell paste, we obtained 9.3 mg of gene 43 protein, 21 mg of gene 45 protein, and 70 mg of a tight complex made up of 44 and 62 proteins; final yields were estimated at 30%, 14%, and 28%, respectively, of the initial activity present in the lysate. When the above purified proteins are incubated with preparations of two other T4 DNA replication proteins (gene 41 and gene 32 proteins) plus deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single- and double-stranded DNA templates. As reported elsewhere, this synthesis mimicks that catalyzed by the T4 DNA replication apparatus in vivo.