Nucleophosmin-1 (NPM1) mutations represent the most frequent gene alteration in acute myeloid leukemia (AML). The most common NPM1 mutation type, accounting for 75 to 80% of cases, is referred to as mutation A (NPM1-mutA). These NPM1 alterations have been shown to possess prognostic significance because they appear to identify patients who will benefit from chemotherapy. Given the high prevalence and stability of these mutations over the course of disease, NPM1 mutations may serve as ideal targets for minimal residual disease (MRD) assessment in AML. Current detection methods are costly, require sophisticated equipment, and are often not sufficiently sensitive. We report here an allele-specific (ASO)-RT-PCR assay that enables rapid and sensitive detection of NPM1-mutA. A semi-nested ASO-PCR method was also designed to increase the sensitivity of our assay for the monitoring of MRD. We analyzed bone marrow cells collected from 52 patients with AML at presentation. NPM1-mutA was detected in leukemic cells from 21 patients. Assay specificity was confirmed by capillary electrophoresis and DNA sequencing. ASO-RT-PCR and semi-nested ASO-PCR assays could detect NPM1-mutA with sensitivities of 10(-2) and 10(-5), respectively. Results obtained here verify that our ASO-RT-PCR assay is a specific and sensitive method for the routine screening of NPM1-mutA, as well as for MRD monitoring of AML patients with this alteration. This method is convenient and easily applicable in countries with limited resources and no access to real-time quantitative PCR-based technologies.