The WT1 gene encodes a transcriptional regulator which during embryogenesis is involved in growth control and differentiation of diverse tissues. It is also expressed in few human malignancies, including acute leukemia. We tested 3 different monoclonal antibodies (MAbs H2, H7, HCl7) and the polyvalent serum WTC-19 for WT1 protein detection in mononuclear cell (MNC) preparations of 104 newly diagnosed acute leukemia patients. Using RT-PCR, these MNC preparations were also analyzed for WT1 gene expression. MAbs H2, H7 and HCl7 and the polyclonal WTC-19 exhibited nuclear immunoreactivity in 63 of 99, 28 of 56, 38 of 60 and 22 of 43 WT1 gene-expressing leukemia samples, respectively. With these antibodies, no WT1 immunoreactivity was found in MNCs from blood of healthy volunteers, from CD34+ progenitor cell-enriched leukapheresis products of patients conditioned for peripheral stem cell harvest or from reactive bone marrow. Contrary to WTC-19, all MAbs reacted highly specifically with the WT1 protein (0.71 vs. 1.0). The WT1 protein was heterogeneously detected in leukemia blast preparations by all antibodies, irrespective of cell morphology. Very few HL60 cells and blasts from newly diagnosed leukemia patients interspersed among normal blood MNCs (50 blasts among 5 x 10(5) MNCs) were easy to identify by indirect immunofluorescence using MAbs H2 and HCl7. Taken together, MAbs H2 and HCl7 were superior to MAb H7 and the polyvalent WTC-19 in detecting the WT1 nuclear protein.