Inv(16)(p13q22) is one of the most frequent chromosomal rearrangements found in acute myelogenous leukemia (AML), representing approximately 16% of documented karyotypic abnormalities. The inv(16) breakpoints have been cloned and shown to involve the non-DNA binding component of the AML-1 transcription factor complex termed core binding factor β gene (CBFβ) on 16q and the smooth muscle myosin heavy chain gene (MYH11) on 16p. In this study, we analyzed 37 cases of inv(16)-containing AML and 4 cases with t(16;16)(p13;q22) for expression of the CBFβ/MYH11 chimeric message by reverse transcription-polymerase chain reaction (PCR) analysis. CBFβ/MYH11 chimeric messages were detected in 33 of 37 cases with the inv(16) and in the 4 t(16;16)-containing cases. Sequence analysis of PCR products showed extensive breakpoint heterogeneity in both CBFβ and MYH11. In addition to the previously described breakpoint in CBFβ at nucleotide (nt) 495 (amino acid 165), we have identified a second novel fusion point at nt 399 (amino acid 133) in 7% of the cases. Similarly, a unique breakpoint site was identified in MYH11 at nt 1098, as well as at three previously characterized sites at nts 994, 1201, and 1921. Of the 4 PCR-negative cases, 2 of 3 tested lacked CBFβ rearrangements by Southern blot analysis, suggesting the possible involvement of a different genomic locus in some cases with cytogenetic evidence of inv(16). To assess whether the portions of CBFβ contained within the CBFβ/MYH11 chimeric products retain the ability to interact with their heterodimeric DNA-binding partner AML-1, we performed in vitro DNA-binding analysis. Recombinant CBFβ polypeptides consisting of the N-terminal 165 amino acids retained their ability to interact with AML-1, whereas mutants containing only the N-terminal 133 amino acids interacted with AML-1 less efficiently. These data suggest that different CBFβ/MYH11 products may vary subtly in their biologic activities.