ZFX, an X chromosome-linked gene encoding a zinc-finger protein, has previously been shown to escape X inactivation in humans. Here we report studies of the inactivation status of the homolog, Zfx, on the mouse X chromosome.We took advantage of both the preferential inactivation of the normal X chromosome in females carrying the T(X;16)16H translocation and the high degree of nucleotide sequence variation between the Mus musculus and Mus spretus genomes. An EcoRV restriction fragment difference between M. musculus and M. spretus was detected after amplification of Zfx transcripts using the reverse transcription-olymerase chain reaction. Using this allelic variation, we assessed expression of the twoZfx genes in females carrying the T(X;16)16H translocation (from M. musculus) and an intact X chromosome (from M. spretus). Such females exhibit Zfx transcription from the active M. musculus chromosome but not from the inactive M. spretus chromosome. These results indicate that the mouse Zfx gene is subject to X inactivation.