Using a two-dimensional gel electrophoresis origin mapping technique and cell synchronization, we have studied replication timing and mapped origins in a 75-kb region of chromosome 2 of Schizosaccharomyces pombe. Three of the five mapped origins are moderately active and the other two are very weak. DNA fragments containing the three moderately active origins and one weak origin are ARS-positive whereas that containing the other weak origin is ARS-negative. Three ARS elements reported earlier from this region appear to be inactive as chromosomal origins. The centromere-proximal 45 kb of this region replicates earlier than the telomere-proximal 30 kb. A transition from early to late replication occurs within 10 kb of the chromosomally inactive ars727, suggesting a possible role of the previously reported late-replication-enforcing region in determining chromosomal replication timing of the region. These results in conjunction with those from some other studies suggest that, in S. pombe, the actual number of potential origins may be significantly higher than previously detected in many genome-wide studies, and the relationship between ARS activity and chromosomal origin activity is not as simple as in Saccharomyces cerevisiae.