Fluorescence in situ hybridization (FISH) is widely used in the study of chromosome structure and organization. Cytogenetic evaluation of chromosomes using FISH technique plays an increasingly important role in diagnosing karyotype changes in both somatic and reproductive cells. The aim of the study was to optimize the conditions of stallion sperm decondensation, which have a significant effect on the results of fluorescence in situ hybridization. Appropriate type and time of decondensation was chosen for the sperm of every stallion. It was found that decondensation performed using a preparation incubated in DTT solution for 1.5 minutes and in SDS solution for 10 seconds proved effective for stallions no. 1 and 2. An alternative decondensation method performed in an Eppendorf tube, with incubation in DTT solution for 1 minute and in SDS solution for 5 seconds proved effective for stallions no. 3 and 4. Decondensation using DTT and papain solution, a method successfully used for bull spermatozoa, proved inadequate for horse spermatozoa.