In female mammals one of the X chromosomes is rendered almost completely transcriptionally inactive to equalize expression of X-linked genes in males and females. The inactive X chromosome is distinguished from its active counterpart by its condensed appearance in interphase nuclei, late replication, altered DNA methylation, hypoacetylation of histone H4, and by transcription of a large cis-acting nuclear RNA called Xist. Although it is believed that the inactivation process involves the association of specific protein(s) with the chromatin of the inactive X, no such proteins have been identified. We discovered a new gene family encoding a core histone which we called macroH2A (mH2A). The amino-terminal third of mH2A proteins is similar to a full-length histone H2A, but the remaining two-thirds is unrelated to any known histones. Here we show that an mH2A1 subtype is preferentially concentrated in the inactive X chromosome of female mammals. Our results link X inactivation with a major alteration of the nucleosome, the primary structural unit of chromatin.