FISH analysis with chromosome painting probes allows, better than karyotyping after Giemsa banding, the study of chromosome segregation after hybridoma formation. FISH is particularly useful for intraspecies hybrids and allows visualization of small chromosome fragments. Cell hybrids were constructed between P3 x 63Ag8.653 mouse myeloma cells and lymphocytes from BALB/c mice by PEG fusion and by selection in hypoxanthine azaserine medium. Three hybridomas (A4, D8, F10) were selected and, after cloning, the cells were cultivated in vitro over a period of 28 days. During this time in culture, air-dried metaphase spreads were prepared by standard methods. For FISH chromosome painting, digoxigenin- and biotin-labeled mouse chromosome painting probes and rhodamine-antidigoxigenin antibodies and fluorescein-avidin were used for dual color detection. Total chromosome numbers and the numbers of mouse chromosomes 1, X, 6 and 12 were estimated as function of days in culture. Mean chromosome numbers of 78 (D8), 82 (F10) and 150 (A4) were observed. The major rearrangements of chromosome numbers occured in the first 28 days in culture and did not change significantly between day 28 and day 56. Mouse chromosome #12, which had the largest chromosome fragments in the parent myeloma, remained stable while the number of X chromosomes, which were significantly fragmented already in the parent myeloma, decreased by approximately 50%.