In yeast, the initiation of DNA replication on chromosomes is believed to occur at specific sequences known as autonomously replicating sequences (ARSs). We previously isolated a number of mutants that are defective in the maintenance of minichromosomes. Analysis of these mutants suggests that although ARSs differ greatly from one another in their primary sequences, they appear to share a common enzyme complex for the initiation of DNA replication. However, this initiation enzyme complex probably binds with differential affinity to different ARSs. This idea is corroborated by our identification of an ARS-binding protein that binds to different ARSs with different efficiencies.