In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a standard DNA preparation – whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsorbed to benzoylated napthoylated DEAE (BND)-cellulose in high salt concentrations. By combining matrix-enrichment and BND-cellulose chromatography, it is possible to obtain preparations that are enriched as much as 200-fold over the starting genomic DNA and are thus suitable for analysis on 2-D gels.