In female mammals one of the X chromosomes is rendered almost completely transcriptionally inactive(1,2) 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(3), late replication(4), altered DNA methylation(2), hypoacetylation of histone H4 (ref. 5), and by transcription of a large cis-acting nuclear RNA called Xist(6-10). 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(11). We discovered a new gene family encoding a core histone which we called macroH2A (mH2A)(12,13). 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.