By means of a cytological technique involving 5-bromodeoxyuridine, acridine orange, and fluorescence microscopy, the asynchronously replicating, hence genetically inactivated, X chromosome was identified in 6- to 8-day embryos from female mice heterozygous for Searle's translocation T(X;16)16H (abbreviated as T16H) mated with either karyotypically normal males or males carrying Cattanach's translocation T(X;7)1Ct in order to analyse the way in which the total inactivation of the normal X is achieved in adult T16H heterozygotes. Embryos examined included 9 Xn/X(7);16/16, 3 X 16/Xn;16x/16, 12 X16/X(7); 16x/16, 5 X16/Xn;16/16, 8 X16/X(7); 16/16 and 2 Xn/Y; 16x/16/16. In these notations X16, 16x, X(7) and Xn represent Searle's X with the centromeric segment of the X, Searle's X with the centomeric segment of chromosome 16, Cattanach's X with insertion of a chromosome 7 segment, and normal X, respectively. The X(7) exerted no apparent effect upon embryonic development up to the 8th day of gestation and X chromosome inactivation. -- The asynchronously replicating X was the Xn in X16/Xn;16x/16 and X(7) in X16/X(7);16x/16 embryos except a small number of cells on day 6 (13/493) and on day 7 (1/886) in which almost the entire 16x replicated asynchronously. The X16, on the other hand, never showed replication asynchrony. That the X16 is indeed unable to become inactivated was indicated by the observation that the X16 as well as Xn or X(7) did not replicate asynchronously in Xn/X16;16/16 and X16/X(7);16/16 embryos X16-inactive cell lines, if occurring, should have been genetically less unbalanced than any other cell line in such embryos. It is highly likely therefore that the ultimate inactivation pattern in T16H heterozygotes has been accomplished by (1) the inability of the X16 to become inactive; (2) inactivation in favor of the Xn; and (3) rapid elimination of 16x-inactive cells. Severe growth retardatin and early death of X16/Xn;16/16 and X16/X(7);16/16 embryos having no inactive X suggested that functional X disomy is detrimental to embryogenesis. These embryos further indicated that the concurrence of at least two X chromosomal loci separated by the T16H breakpoint is necessary for the homologous X chromosome becoming inactivated.