X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XYmales and XXfemales. However, it has never beenextensively studied ina eutherian species witha sex determination system thatdeviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouseMusminutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, namedX*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modi-fication patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated inXX* females, while no histone modifications were detected in X*Y females. Furthermore, inM. minutoides, X and X*chromosomes are fused todifferent autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished

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• X inactivation in a mammal species with three sex chromosomomes