Replication of eukaryotic chromosomes involves initiation at origins spaced an average of 50 to 100 kilobase pairs.In yeast, potential origins can be recognized as autonomous replication sequences (ARSs) that allow maintenance of plasmids. However, there are more ARS elements than active chromosomal origins. The possibility was examined that close spacing of ARSs can lead to inactive origins. Two ARSs located 6.5 kilobase pairs apart can indeed interfere with each other. Replication is initiated from one or the other ARS with equal probability, but rarely ("less than"5%) from both ARSs on the same DNA molecule.