Cytogenetic analysis using banding techniques of B-chronic lymphocytic leukemia (CLL) is hampered by the difficult in vitro proliferation of these tumor cells. For detection of specific cytogenetic aberrations these problems can be overcome with non-radioactive in situ hybridization (ISH). ISH may especially be applied for the detection of trisomy 12, which is the most frequent cytogenetic aberration in CLL. Sixty-seven patients with CLL, four normal controls and one lymphoblastoid B-cell line with a trisomy 12 were studied using a chromosome 12 specific probe. To determine the hybridization properties of the CLL cells, all samples were also hybridized with probes specific for chromosomes 1 and 8. All leukemias were analyzed by immunocytochemistry to determine the proportion of tumor cells. Eight cases (11%) showed a trisomy 12. After correction for the number of tumor cells, it was demonstrated that in almost all cases (7 out of 8), the aberration was present in a proportion of the tumor cells (between 30 and 72%). Except for one patient this mosaicism persisted with long-term follow-up. We conclude that the in vivo incidence of trisomy 12 in CLL is approximately 11%, and that trisomy 12 occurs in most instances in only a subpopulation of the leukemic cells. Both findings suggest that trisomy 12 in CLL is a late event.