Patients in accelerated phase or blast crisis of chronic myeloid leukemia (CML) frequently develop clonal cytogenetic abnormalities in addition to the Philadelphia chromosome. Using a DNA probe directed to the centromere of chromosome 8, we performed fluorescence in situ hybridization (FISH) on archival Wright-stained blood and bone marrow smears of seven patients with CML and with a known +8 clone by metaphase cytogenetics to determine the distribution of +8 in interphase cells. All slides had been stored at ambient temperature for 12-26 months. The bone marrow aspirate smears of 21 non-leukemic patients served as controls. Trisomy 8 was demonstrated in all myeloid cell lines including the neutrophils, basophils, eosinophils, monocytes, and erythroid precursors, but not in the lymphocytes. The extra chromosome 8 was present in mature segmented granulocytes as well as more immature precursors. The percentage of +8 cells was highest in specimens from patients with CML in myeloid blast crisis (mean 64%), followed by those in accelerated phase (mean 39%). Three specimens from patients in morphologic chronic phase showed the lowest percentage of +8 cells (mean 13%). One patient was studied twice and showed a substantial expansion of +8 cells with progression from accelerated phase to myeloid blast crisis. Compared to metaphase cytogenetics, the proportion of +8 cells detected by FISH was often lower. We conclude that the acquisition of trisomy 8 in CML occurs in a pluripotent myeloid stem cell apparently incapable of expressing mature lymphoid phenotype, and that morphologic progression of disease is generally associated with an expansion of the +8 component.