In mammals, gene dosage of X-chromosomal genes is equalized between the sexes by random inactivation of either one of the two X chromosomes in female cells. Xist, Tsix and Xite are non-coding genes located in a small region on the X chromosome, which play a crucial role in X-chromosome inactivation (XCI). Xist RNA accumulates on the future inactive X chromosome and initiates silencing in cis. Tsix and Xite down-regulate Xist transcription, acting against XCI. In the initial phase of XCI, a counting and initiation process determines the number of X chromosomes per nucleus, and elects the future inactive X chromosome. We found that this process is directed by a stochastic mechanism, in which each X chromosome has a specific probability to be inactivated. We also showed that the probability to initiate XCI is determined by the X:ploïdy ratio. These results indicated the presence of at least one X-linked activator of the XCI process. With a BAC screen we recently identified X-encoded RNF12 to be an activator of XCI. A heterozygous deletion of Rnf12 results in a marked loss of XCI in female cells, confirming a prominent role of RNF12 in XCI. The remained presence of a small proportion of cells that do initiate XCI also indicated that more XCI-activators are involved in XCI. Here, we propose to investigate the molecular mechanism by which RNF12 activates XCI, and to search for additional XCI-activators. Complementary to this, we will also attempt to determine the structure of Xist RNA and its RNA-protein complexes. In addition, we aim to determine the role of RNF12 and other XCI-activators in human XCI. We anticipate that these studies will significantly advance our understanding of XCI mechanisms, which is highly relevant for a better insight in the manifestation of X-linked diseases that are affected by XCI.