Replication of the human genome requires the activation of thousands of replicons distributed along each one of the chromosomes. Each replicon contains an initiation, or origin, site, at which DNA synthesis begins. However, very little information is known about the nature and positioning of these initiation sites along human chromosomes. We have recently focused our attention to a 1.1 kb region of human chromosome 2 which functioned as an episomal origin in the yeast Saccharomyces cerevisiae. This region corresponded to the largest exon of a putative ribulose-5-phosphate-3-epimerase gene (RPE). In the present study we have used a real-time PCR-based nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 13.4 kb region encompassing the putative RPE gene. By applying this analysis to a 1-1.4 kb nascent strand DNA fraction isolated from both normal skin fibroblasts, and the breast cell line MCF10; we have identified five initiation sites within the 13.4 kb region of chromosome 2. The initiation sites appear to map to similar positions in both cell lines and occur outside the coding regions of the putative RPE gene. (C) 2004 Elsevier Inc. All rights reserved.