Nucleophosmin 1 (NPM1) is a nuclear protein and in approximately 50% to 60% of cytogenetically normal acute myeloid leukemia (AML), NPM1 is mutated and localized in the cytoplasm. Both wild type and mutant NPM1 can be detected by immunohistochemistry (IHC). We set out to evaluate whether subcellular localization of NPM1, as detected by IHC, is stable during disease course in AML, and whether cytoplasmic expression of NPM1 (NPMc+) can be used to differentiate recovery versus residual disease in formalin-fixed and hydrochloric acid/EDTA decalcified specimens. IHC against NPM1 was performed on bone marrow biopsies of 31 patients with AML at initial diagnosis and on 40 follow-up biopsies. Immunostaining patterns of NPM1, defined as NPMc+, NPMc-, and indeterminate, were correlated with morphology, ancillary tests, and clinical follow-up information. Of the 20 (64.5%) cases with NPMc- at initial diagnosis, none of the 27 follow-up biopsies showed NPMc+, regardless of disease status. Of the 11 cases (35.5%) with NPMc+ at initial diagnosis, all of the 3 biopsies with persistent/relapsed disease, and none of the 7 benign biopsies had NPMc+ at follow-up. For the 3 biopsies with indeterminate follow-up pathologic diagnoses of recovery versus residual disease, all were NPMc- at follow-up, which was consistent with remission and was supported by clinical follow-up. Therefore, subcellular localization of NPM1 as detected by IHC is stable during the course of the disease, and NPMc+ at follow-up supports residual disease in cases with NPMc+ at initial diagnosis.