DNA replication origins (ORIs) map close to promoter regions in many organisms, including mammals. However, the relationship between initiation of replication and transcription is not well understood. To address this issue, we have analyzed replication timing and activity of several CpG island-associated ORIs on the transcriptionally active and silent X chromosomes. We find equivalent ORI usage and efficiency of both alleles at sites that are replicated late on the inactive X chromosome. Thus, in contrast to its repressive effect on transcription, heterochromatin does not influence ORI activity. These findings suggest that the relationship between sites of transcription and replication initiation at CpG island regions is restricted to early development, and that subsequent gene silencing and heterochromatin formation influence only the timing of ORI activation.