DNA replication in metazoans initiates from multiple chromosomal loci called origins. Currently, there are two methods to purify origin-centered nascent strands: lambda exonuclease digestion and anti-bromodeoxyuridine immunoprecipitation. Because both methods have unique strengths and limitations, we purified nascent strands by both methods, hybridized them independently to tiling arrays (1% genome) and compared the data to have an accurate view of genome-wide origin distribution. By this criterion, we identified 150 new origins that were reproducible across the methods. Examination of a subset of these origins by chromatin immunoprecipitation against origin recognition complex (ORC) subunits 2 and 3 showed 93% of initiation peaks to localize at/within 1 kb of ORC binding sites. Correlation of origins with functional elements of the genome revealed origin activity to be significantly enriched around transcription start sites (TSSs). Consistent with proximity to TSSs, we found a third of initiation events to occur at or near the RNA polymerase II binding sites. Interestingly, ∼50% of the early origin activity was localized within 5 kb of transcription regulatory factor binding region clusters. The chromatin signatures around the origins were enriched in H3K4-(di- and tri)-methylation and H3 acetylation modifications on histones. Affinity of origins for open chromatin was also reiterated by their proximity to DNAse I-hypersensitive sites. Replication initiation peaks were AT rich, and >50% of the origins mapped to evolutionarily conserved regions of the genome. In summary, these findings indicate that replication initiation is influenced by transcription initiation and regulation as well as chromatin structure. © 2010 by The American Society for Cell Biology.