Cytosine methylation at promoter regions and late replication timing have both been implicated in the regulation of genes subject to X chromosome inactivation. Reported here are studies of X-linked gene replication in normal male and female cells as well as in cell hybrids that contain either a normal active X, a normal inactive X, or an inactive X chromosome that has been treated with the demethylating agent, 5-azacytidine (5aC). The relationship between replication timing and transcriptional activity was examined for XIST, XPCT, PGK1, HPRT, F9, FMR1, IDS, and G6PD, and earlier replication was generally found to be associated with increased transcriptional activity. The HPRT and G6PD genes in an untreated inactive X hybrid were among the few exceptions to this correlation in that they remain inactive, yet replicate earlier than their inactive X alleles present in normal human diploid cells. This condition of earlier replication timing may contribute to the high rates of 5aC-induced reactivation for HPRT and G6PD in this hybrid relative to other inactive X hybrids. Other anomalous cases include 5aC-induced advances in replication time for genes such as XIST and F9 whose transcription was unaltered by treatment. These and other data support a model for regulation of X-inactivated genes that involves at least two levels of control: (i) large chromosomal domains are placed into a transcriptionally nonpermissive state by late replication and (ii) transcription is blocked at the local level by promoter methylation. In addition, our observations of continued XIST expression in 5aC-treated hybrids with reactivated genes indicates that such expression is not sufficient for the maintenance of X inactivation.