Mutations in the nucleophosmin (NPM1) exon 12 resulting in delocalization of NPM1 into the cytoplasm occur in 50% to 60% of acute myeloid leukemia cases with a normal karyotype (AML-NK). As recent studies suggest such patients have a favorable prognosis and there are discordant reports of the immunohistochemical detection of cytoplasmic NPM1 (NPMc+) for predicting NPM1 gene mutations, we correlated the immunohistochemical detection of NPMc+, NPM1 gene mutations, and prognosis in 57 cases of AML-NK. All 31 NPMc+ cases (54% of total) had NPM1 mutations, but none of the 26 nucleus-restricted (NPMc-) cases (46% of total) had NPM1 mutations (P < .0001). NPM1 mutations were correlated with FLT3-internal tandem duplication (ITD) (P = .0062), absence of CD34 (P = .0001), and absence of CD7 (P = .041). There was a favorable survival outcome in AML-NK cases that were NPM1 mutated and FLT3-ITD nonmutated. Our data confirm that cytoplasmic NPM1 immunoreactivity predicts NPM1 mutations and warrants inclusion in the routine diagnostic and prognostic workup of AML.