Acute myeloid leukemia with inv(16)(p13q22), also known as M4Eo, is a distinct type of leukemia with characteristic clinicopathologic and cytogenetic features. Patients with M4Eo have monocytosis, high blast counts, and abnormal bone marrow eosinophils that contain large basophilic granules. The inv(16)(p13q22) or, less commonly, the t(16;16)(p13;q22) causes fusion of the CBFbeta gene at 16q22 and the MYH11 gene at 16p13, creating the novel chimeric protein CBFbeta-MYH11. To understand the underlying molecular mechanisms unique to M4Eo biology, we determined the gene expression profile of M4Eo cases by using cDNA and long oligonucleotide microarrays. Cases of acute myelomonocytic leukemia without CBFbeta-MYH11 (M4) acted as our control. We found that in the gene expression profile of M4Eo, NF-kappaB activators and inhibitors were upregulated and downregulated, respectively, suggesting that the NF-kappaB signaling pathway is activated at a higher level in M4Eo than in acute myelomonocytic leukemia M4. In addition, the gene expression profile of M4Eo indicates high cell proliferation and low apoptosis. We used real-time PCR, immunohistochemistry, and flow cytometry immunophenotyping to confirm some of our microarray data. These findings most likely represent the functional consequences of the abnormal chimeric protein CBFbeta-MYH11, which is unique to this disease, and suggest that NF-kappaB is a potential therapeutic target for treating M4Eo patients.