We have identified five autonomously replicating sequences (ARSs) in a 100 kbp region of the Schizosaccharomyces pombe chromosome II. Analyses of replicative intermediates of the chromosome DNA by neutral/neutral two-dimensional gel electrophoresis demonstrated that at least three of these ARS loci operate as chromosomal replication origins. One of the loci,ori2004, was utilized in almost every cell cycle, while the others were used less frequently. The frequency of initiation from the respective chromosomal replication origin was found to be roughly proportional to the efficiency of autonomous replication of the corresponding ARS plasmid. Replication from ori2004 was initiated within a distinct region almost the same as that for replication of the ARS plasmid. These results showed that the ori2004 region of approximately 3 kbp contains all the cis elements essential for initiation of chromosome replication.