Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.