Antigen-specific rat mouse T-cell hybridomas, prepared by fusing the BW5147 AKR thynoma with helper T cells from rats immunized with chicken ovalbumin, express both the rat and mouse major histocompatibility complex gene products. Using these functionally defined T-cell hybridomas, we have devised a technique based on the differential staining of the heterochromatin region of the rat and mouse chromosomes to distinguish the species of origin of the chromosomes. This technique should prove useful for screening potential hybrid cell lines and should aid in differentiating mouse from rat chromosomes for gene mapping studies.