While the distribution of RNA polymerase II (PoIII) in a variety of complex genomes is correlated with gene expression, the presence of PoIII at a gene does not necessarily indicate active expression. Various patterns of PoIII binding have been described genome wide; however, whether or not PoIII binds at transcriptionally inactive sites remains uncertain. The two X chromosomes in female cells in mammals present an opportunity to examine each of the two alleles of a given locus in both active and inactive states, depending on which X chromosome is silenced by X chromosome inactivation. Here, we investigated PoIII occupancy and expression of the associated genes across the active (Xa) and inactive (Xi) X chromosomes in human female cells to elucidate the relationship of gene expression and PoIII binding. We find that, while PoIII in the pseudoautosomal region occupies both chromosomes at similar levels, it is significantly biased toward the Xa throughout the rest of the chromosome. The general paucity of PoIII on the Xi notwithstanding, detectable (albeit significantly reduced) binding can be observed, especially on the evolutionarily younger short arm of the X. PoIII levels at genes that escape inactivation correlate with the levels of their expression; however, additional PoIII sites can be found at apparently silenced regions, suggesting the possibility of a subset of genes on the Xi that are poised for expression. Consistent with this hypothesis, we show that a high proportion of genes associated with PoIII-accessible sites, while silenced in GM12878, are expressed in other female cell lines. © The Author 2011. Published by Oxford University Press. All rights reserved.