Nucleophosmin (NPM) is a nucleus-cytoplasmic shuttling protein that is implicated in centrosome duplication, cell cycle progression and stress response. At the steady state, NPM localizes mainly in the nucleolus, where it forms a complex with different cellular proteins. One-third of acute myeloid leukemias (AML) are characterized by aberrant cytoplasmic localization of NPM, due to mutations within its last coding exon (exon 12) that cause a frameshift and the formation of novel C-termini. We report here our investigations on the molecular basis for the aberrant localization of mutated NPM. Alignment of the C-terminus of the various NPM mutants revealed the obligatory presence of four amino-acid residues that match a CRM1-dependent nuclear export signal (NES). Single alanine-substitutions at these sites provoked nuclear re-localization, while fusion of the mutated C-terminus to a heterologous nuclear protein induced CRM1-dependent cytoplasmic localization. Molecular characterization of one exceptional AML carrying cytoplasmic NPM and germ line exon 12 revealed a somatic mutation in the splicing donor site of exon 9 that caused the formation of a functional NES. It appears, therefore, that AMLs are frequently characterized by gain-of-function mutations of NPM that create functional NES, suggesting that alterations of nuclear export might represent a general mechanism of leukemogenesis and a novel target for therapeutic intervention.