Two leukemia cell lines, TS9;22 and YS9;22, were established from different individuals with Philadelphia chromosome (Ph)-positive chronic myeloid leukemia in blast crisis. The reverse transcript-polymerase chain reaction (RT-PCR) technique revealed that both cell lines expressed GATA-1, GATA-2, and the stem cell leukemia (SCL) gene, consistent with a megakaryocyte lineage. Chromosome analysis revealed that TS9;22 cells show the Ph translocation without abnormality of chromosome 3. In contrast, YS9;22 cells show the Ph translocation and dic(3)(q26;p12). Northern analysis revealed that YS9;22 cells express the EVI1 (ecotropic virus integration-1) gene, possibly because of the chromosomal translocation in the 3q26 region; TS9;22 cells do not express EVI1. However, no rearrangements were detected over 600 kb upstream or over 900 kb downstream of EVI1 in the YS9;22 cell line, suggesting a different mechanism of EVI1 activation from that in leukemia cells with either a t(3;3)(q21;q26) or inv(3)(q21q26). These results indicate that EVI1 expression in YS9;22 cells is linked to the 3q26 abnormality and that EVI1 activation plays an oncogenic role in the blastic transformation of chronic myeloid leukemia.