pANCA is a marker antibody associated with inflammatory bowel disease (IBD), including most patients with ulcerative colitis and a subset with Crohn's disease. This study addressed the hypothesis that pANCA reacts with an antigen(s) of microbial agents potentially relevant to IBD pathogenesis. Using a pANCA monoclonal antibody, we have previously identified the C-terminal basic random-coil domain of histone H1 as a pANCA autoantigen. BLAST analysis of the peptide databases revealed H1 epitope homologues in open reading frames of the Mycobacterium tuberculosis genome. Western analysis of extracts from six mycobacterial species directly demonstrated reactivity to a single, conserved approximately 32-kDa protein. Direct protein sequencing, followed by gene cloning, revealed a novel 214-amino-acid protein, an iron-regulated protein recently termed HupB. Sequence analysis demonstrated its homology with the mammalian histone H1 gene family, and recombinant protein expression confirmed its reactivity with the 5-3 pANCA monoclonal antibody. Binding activity of patient serum immunoglobulin G (IgG) to HupB did not correlate with reactivity to histone H1 or pANCA, indicating the complex character of the pANCA antigen. However, anti-HupB IgA was strongly associated with Crohn's disease (P < 0.001). These findings indicate that the 5-3 pANCA monoclonal antibody detects a structural domain recurrent among mycobacteria and cross-reactive with a DNA-binding domain of histone H1. The association of HupB-binding serum IgA with IBD provides new evidence for the association of a mycobacterial species with Crohn's disease.