Approximately one third of acute myeloid leukemias (AMLs) are characterized by aberrant cytoplasmic localization of nucleophosmin (NPMc⁺ AML), consequent to mutations in the NPM putative nucleolar localization signal. These events are mutually exclusive with the major AML-associated chromosomal rearrangements, and are frequently associated with normal karyotype, FLT3 mutations, and multilineage involvement. We report the gene expression profiles of 78 de novo AMLs (72 with normal karyotype; 6 without major chromosomal abnormalities) that were characterized for the subcellular localization and mutation status of NPM. Unsupervised clustering clearly separated NPMc⁺ from NPMc^– AMLs, regardless of the presence of FLT3 mutations or non–major chromosomal rearrangements, supporting the concept that NPMc⁺ AML represents a distinct entity. The molecular signature of NPMc⁺ AML includes up-regulation of several genes putatively involved in the maintenance of a stem-cell phenotype, suggesting that NPMc⁺ AML may derive from a multipotent hematopoietic progenitor.