MacroH2A1 is an unusual variant of the core histone H2A which is enriched in chromatin on the inactive X chromosome of female mammals. The N-terminal third of the protein shares 65% amino acid identity with core histone H2A, while the remaining two-thirds of the protein are novel, with a small stretch of basic amino acids and a putative leucine zipper motif. We have now cloned a second macroH2A gene, encoding macroH2A2 which shares 80% amino acid identity with macroH2A1. Despite mapping to different chromosomes, the genomic organization of the macroH2A2 and macroH2A1 genes are nearly identical. The leucine zipper motif of macroH2A1 is not conserved in macroH2A2. Like macroH2A1, macroH2A2 forms a Macro Chromatin Body in the nuclei of female cells which is coincident with an X chromosome and co-localizes with macroH2A1. To address the distribution of other histone H2A variants in relation to macroH2A and the inactive X chromosome, we constructed a series of epitope-tagged versions of other histone H2A variants. Like the recently described H2A-Bbd (Barr body-deficient) variant, the histone variant H2A.Z was found to be deficient in chromatin on the inactive X chromosome in a significant proportion of female nuclei. This study provides further information about the nucleosomal composition of chromatin on the inactive X chromosome and indicates that a number of H2A variants are non-randomly distributed on the active and inactive X chromosomes.