The specific (6;9)(p23;q34) chromosomal translocation is associated with a defined subtype of acute nonlymphocytic leukemia (ANLL). The 9q34 breakpoint is located at the telomeric side of the c-abl gene. Through a combination of chromosome jumping, long-range mapping, and chromosome walking, the chromosome 9 breakpoints of several t(6;9) ANLL patients were localized within a defined region of 8 kilobases (kb), 360 kb telomeric of c-abl. Subsequent cDNA cloning revealed that this region represented an intron in the middle of a gene, called Cain (can), encoding a 7.5-kb transcript. Disruption of the can gene by the translocation resulted in the expression of a new 5.5-kb can mRNA from the 6p- chromosome. Isolation of chromosome 6 sequences showed that breakpoints on 6p23 also clustered within a limited stretch of DNA. These data strongly suggest a direct involvement of the translocation in the leukemic process of t(6;9) ANLL.