Eukaryotic DNA replication initiates at multiple points along the chromosomes known as replication origins (ORIs). We have developed a strategy to identify ORIs directly from replication intermediates in the fission yeast Schizosaccharomyces pombe. Mapping of a selection of the novel ORIs onto the genome reveals their preferential localization at intergenic regions upstream from genes. These results are supported by the observation that a large proportion of regions overlapping gene promoters contain active ORIs. Mapping of the genomic ars1 replication origin at nucleotide resolution shows that replication initiates at a defined position immediately upstream from the hus5(+) promoter. Deletion analysis indicates that the regulatory elements required to initiate transcription and replication lie in close proximity, suggesting a possible relationship between both processes in vivo.