In a series of six independent experiments organ homogenates of 35 mice of the XX, XO or XY sex chromosome constitutions were absorbed using three different anti-H-Y antisera raised in inbred female LEW rats. Residual activities of absorbed antisera were tested in the Raji cell, complement-dependent, cytotoxicity test. Homogenates of various tissues, including the gonads, of XX and XO females were equally unable to absorb H-Y antibodies, indicating that tissues of these mice do not carry the H-Y antigen. In contrast, XY male homogenates fully absorbed H-Y antibodies of antisera at concentrations of 1/2 to 1/4. We discuss our findings with special attention to the problem of the existence of one or more H-Y antigens and, to the genetic regulation of the expression of this antigen.