Cellular protein Naf1 (Nef-associated factor 1) or ABIN-1 (A20-binding inhibitor of NF-kappaB activation) is an important cellular protein, expressed in various human tissues and T-cell lines. Naf1 protein has two isoforms (Naf1alpha and Naf1beta) with different C-termini, produced by alternative splicing. Naf1alpha and Naf1beta have approximately 2800 and 2600 nucleotides, with an open reading frame of 1941 and 1781 nucleotides, encoding the 72-kDa Naf1alpha and 68-kDa Naf1beta proteins, respectively. In the present study, we generated a monoclonal antibody (MAb) against human Naf1, which recognizes full-length, endogenous Naf1 of both isotypes. For this purpose, recombinant 6xHis and myc-tagged N-terminal Naf1(38135), Naf1(N) protein was produced by using the baculovirus expression system. Recombinant Naf1(N) protein was used to immunize Balb/c mice, and a hybridoma cell line producing stable and highly specific MAb with strong affinity to Naf1 was established. We further characterized this antibody by immunofluorescent assay and Western blot analysis to confirm effectiveness in detecting recombinant and endogenous Naf1. By Western blot analysis of recombinant Naf1-N fusion proteins with overlapping N-terminal sequences, the epitope targeted by anti-Naf1 MAb was determined as the 81-88-amino acid region of human Naf1.