Naf1 (Nef-associated factor 1)/TNIP1/ABIN-1 (A20-binding inhibitor of NF-κB activation) is a cellular protein that interacts and cooperates with the NFκB inhibiting protein A20. It is reported that Naf1 attenuates epidermal growth factor (EGF)/extracellular-signal-regulated kinase2 (ERK2) nuclear signaling. Naf1 also binds to Nef, which plays a key role in acquired immunodeficiency syndrome pathogenesis and HIV-1 virus replication. Naf1 mRNA consists of 18 exons and multiple splice variants have been reported; two isoforms for exon 1, deletion of exon 2 and isoforms α and β for exon 18. Using specimens from 29 acute myeloid leukemia (AML) patients, we detected a high frequency of allelic loss on DNA at STS marker D5S2014 near the Naf1 gene. We therefore performed mutation and expression analyses using leukemia-lymphoma lines and 6 pairs of clinical AML samples. There was no mutation in the Naf1 coding region of any sample. As a result of expression analysis, we identified novel splice variants of the Naf1 gene; deletion of exon 16 (Naf1 α2, Naf1 β2), deletion of exon 16 with an insertion (Naf1 α3, Naf1 β3) and deletion of exons 16 and 17 (Naf1 α4). Naf1 α3 and β3 showed premature termination. In peripheral blood mononucleocytes (PBMNCs) from healthy adults, almost no expression of full-length Naf1 (Naf1FL), Naf1 α3 and β3 were observed. In contrast, their expression was clear in AML blasts and in the majority of leukemia-lymphoma lines investigated. Naf1 α2 was widely expressed in PBMNCs from healthy adults, AML blasts and cell lines, suggesting it is the main transcript of the Naf1 gene. Luciferase assay revealed that Naf1 α2 had equal NF-κB inhibitory effect to that of Naf1FL, while Naf1 α4 was less effective. In clinical AML patients, the expression of Naf1 α3 was much higher at diagnosis than on remission after chemotherapy, suggesting the possible dominant negative effect of Naf1 α3.