For the cytogenetic study of X chromosome inactivation as an X chromosome dosage compensation mechanism, we isolated a number of XXXX, XXX, and XXY near-tetraploid mouse hybrid cell clones by fusing XX or XO embryonal carcinoma cells with lymphocytes carrying a structurally altered X chromosome(s). The inactive X chromosome from the female lymphocyte was reactivated in these hybrid clones which retained embryonal carcinoma morphology so far as they were cultured on the collagen-coated plastic surface in the medium supplemented with leukemia inhibitory factor (LIF) and betamercaptoethanol (BME). Some of these clones developed balloon-like cystic embryoid bodies when they were allowed to form cell aggregates in medium without LIF and BME in bacteriological petri dishes to which they do not adhere. X chromosome inactivation occurring during this process detected by the incorporation of 5-bromodeoxyuridine did not conform to the expected pattern leaving two X chromosomes active in every tetraploid cells. This may suggest either that the X-inactivation mechanism evolved primarily, for the diploid cell is unable to deal with tetraploid conditions efficiently, or that the present system of in vitro differentiation represents an anomalous situation never encountered in vivo.