AbstractDNA fragments that contain an active origin of replication generate bubble‐shaped replication intermediates with diverging forks. We describe two methods that use two‐dimensional (2‐D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificialSaccharomyces cerevisiaechromosomes. The first method uses 2‐D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2‐D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restri