Detailed procedures are presented which allow reproducible preparation of T4 gene 32 protein, a helix-destabilizing protein essential for DNA replication and genetic recombination in T4 bacteriophage-infected Escherichia coli cells. Although 32 protein can be purified to better than 99% homogeneity by any one of several procedures, these methods have been developed to remove trace amounts of contaminating deoxyribonucleases, which are present in high levels in the original infected cells. Two alternative preparations are presented, each involving three chromatographic steps. Both 32 proteins obtained are essentially "nuclease-free," when tested at physiological salt concentrations. However, we show here that the phenyl-Sepharose chromatography step, which is necessary to remove an exonuclease activity active only at low salt concentrations, also removes a second protein present in trace amounts. In some cases, retention of this second protein is desirable, since it is essential for obtaining RNA primed, de novo DNA chain starts in an in vitro DNA replication system, when this system is constructed by mixing highly purified preparations of each of the six replication proteins coded for by T4 genes 32, 43, 44, 62, 45, and 41.