Inversion 16 (Inv(16)) results in a fusion between genes CBFB on the q arm and MYH11 on the p arm and is associated with acute myeloid leukemia (AML) subtype M4Eo. CBFB encodes a protein (CBFb) that forms a heterodimeric transcription factor with RUNX1/AML1, which itself is disrupted by several chromosome translocations in leukemias. MYH11 encodes the smooth muscle myosin heavy chain (SMMHC). The fusion gene CBFB–MYH11, through its encoded CBFβ–SMMHC fusion protein, dominant negatively disrupts the normal function of the CBFβ/AML1 dimer in gene expression regulation and hematopoiesis. Using cDNA microarray technology we attempted to identify gene expression profiles characteristic to Inv(16) in comparison with non-Inv(16) AML and normal bone marrow (BM). RNA from five cases of M4Eo AML with Inv(16), seven cases with M4 AML without inv(16) and five normal BM samples was used to generate labeled cDNA probes to hybridize against a 6.7-kD human cDNA microarray. We used a common reference probe generated from the cell line HL-60 to co-hybridize with cDNA probes derived from patients, allowing normalization of the expression level and ratio of expression of each gene in each sample relative to HL-60. M4Eo, M4 and normal BM were the principal three clusters or groups of samples observed when the expression ratios were compared using hierarchical clustering and multidimensional scaling (MDS). The maximum difference in gene expression profiles as computed by MDS and assigned as a measure of distance between samples was that for M4Eo and M4 versus normal BM. We have generated a list of weighted genes whose expression differences have the greatest power to distinguish among the groups. Those gene expression changes that are unique to the Inv(16) cases will be studied further. This study may help us better understand the oncogenic mechanism of CBFB–MYH11 and provide new therapeutic targets.