The results of in situ hybridization with labeled species specific and X-chromosome-specific probes suggest that hybrid cells obtained by fusion of Mus musculus embryonic stem cells (genotype XY) and splenocytes of M. caroli females contain two parental X-chromosomes. In five clones of hybrid cells, differentiation was induced in embryoid bodies in vitro, which was accompanied by inactivation of one of X-chromosomes. We analyzed the expression of Xist and Gla alleles in the embryoid bodies using RT-PCR with an account that expression of locus Xist is one of key events in X-chromosome inactivation, while gene Gla was used as a marker of active X-chromosome. Identification of allele transcripts of loci Xist and Gla was based on restriction polymorphism between M. musculus and M. caroli that we had described. Transcripts of both parental alleles of loci Xist and Gla were present in the embryoid bodies of all studied hybrid clones. No preferential inactivation of M. musculus or M. caroli X-chromosome was found in the tested embryonic hybrid cells despite the initial differences in ontogenetic status between X-chromosomes of embryonic stem cells and splenocytes.