The aim of the study was to optimize hybridization conditions of molecular probes specific for X sex chromosomes of the domestic horse in mare oocyte chromosomes. Mare oocytes, recovered from slaughterhouse ovaries by scraping the granulosa layer, were cultured in vitro. Metaphase II mature oocytes were treated with hypotonic solution and fixed, followed by hybridization of themolecular probe specific for the X chromosome of the domestic horse. Hybridization of probes specific formouse heterosomes onmouse oocytes and early embryos was performed to verify the FISH technique. Of 438 oocytes analysed, 29% reached metaphase II. Despite many changes in the composition of hypotonic solutions and modification of the FISH protocol, the fluorescence signal was observed in mouse oocytes and embryos but not in mare oocytes.