Specific rearrangements involving 3q21 and 3q26 are well documented in acute myeloid leukemia (AML). Aberrant expression of the Ecotropic virus integration-1 (EVI1) gene, located at 3q26, has been reported in individuals with AML and translocations or inversions of chromosome 3 long arm. We have studied six individuals with AML and inv(3)(q21q26) for disruptions to the EVI1 locus by in situ hybridization and long-range mapping. EVI1 transcripts have been detected in the blast cells of the two individuals available for expression studies. We derived a YAC containing the EVI1 gene and showed that it crossed the 3q26 inversion breakpoints in three of four cases examined. Pulsed field analysis detected aberrant fragments 3' of the EVI1 gene in all six patients. The orientation of the gene was established and the locations of the breakpoints were refined by in situ hybridization using phage clones from this region.