AbstractWe previously characterized a functional origin of DNA replication at the transcriptional promoter of the human hypoxanthine‐guanine phosphoribosyltransferase (HPRT) gene (Cohen et al. [2002] J. Cell. Biochem. 85:346‐356). This origin was mapped using a quantitative PCR assay to evaluate the relative abundance of HPRT markers in short nascent DNA strands isolated from asynchronous cultures of male fibroblasts. The HPRT gene on the X chromosome is transcriptionally active in male human fibroblasts. It is known that on the heterochromatic X chromosome in female cells the HPRT gene is transcriptionally silenced and its replication timing changes from early to late in S phase. This change in replication timing could indicate that replication of the HPRT gene is under the control of different origins of DNA replication in the active (euchromatic, early replicating) and the inactive (heterochromatic, late replicating) X chromosomes. In the present study, we identified the location of the origin of replication of a second X chromosome gene, glucose‐6‐phosphate dehydrogenase (G6PD), which we mapped to its transcriptional promoter, in normal male human fibroblasts. Then, we determined the activity of the previously identified HPRT and the G6PD human origins in hybrid hamster cells carrying either the active or the inactive human X chromosome. The results of these studies clearly demonstrated that the human HPRT and G6PD origins of replication were utilized to the same extent in the active and the inactive X chromosomes. Therefore, transcription activity at the HPRT and G6PD genes is not necessary for initiation of DNA replication at the origins mapped to these chromosomal loci. J. Cell. Biochem. 88: 923–931, 2003. © 2003 Wiley‐Liss, Inc.