AbstractX‐chromosome activity in female mouse embryos was studied at the cellular level using an X‐linked lacZ transgene which encodes β‐galactosidase (β‐Gal). Translation of maternal RNA in oocytes is seen as β‐Gal activity that persists into early cleavage‐stages. Zygotic transcription of the transgene from the maternal X chromosome (Xm) is first found at about the 8‐cell stage. By contrast, expression of the lacZ transgene on the paternal X chromosome (Xp) is not seen until later at the 16‐32‐cell stage. Preferential inactivation of Xp occurs in the mural trophectoderm, the primitive endoderm, and derivatives of the polar trophectoderm, but a small number of cells in these lineages may still retain an active paternal X chromosome. X inactivation begins at 3.5 days in the inner cell mass but contrary to previous findings the process is not completed in the embryonic ectoderm by 5.5 to 6.0 days. Regional variation in β‐Gal activity is also observed in the embryonic ectoderm during gastrulation which may be related to the specification of cell fates. Random inactivation of Xp and Xm ensues in all somatic tissues but the process is completed at different times in different tissues. The slower progression of X inactivation in tissues such as the notochord, the heart, and the embryonic gut is primarily due to the persistent maintenance of two active X chromosomes in a significant fraction of cells in these tissues. Recent findings on the methylation of endogenous X‐linked genes suggest that the prolonged expression of β‐Gal might also be due to the different rate of spreading of inactivation along the X chromosome to the lacZ transgene locus in different tissues. © 1994 Wiley‐Liss, Inc.