Levels of specific salivary IgA antibodies against mycobacterial heat shock protein (hsp) 65 are significantly increased in patients with gingivitis when compared to clinically healthy subjects. The process of identifying the hsp65 epitopes recognized by the salivary antibodies, binding to overlapping 15-mer-hsp65 peptides, was assessed. Time-resolved fluorescence immunoassays using 15-mer overlapping peptides spanning the whole hsp65 molecule revealed six distinct sequences recognized by anti-hsp65 IgA antibodies. Due to the high degree of sequence homology between mycobacterial hsp65, cognates of the hsp60 family of oral bacterial flora and human hsp60, these six epitopes may serve as cross-reactive autoantigens in certain circumstances in vivo and could incite an autoimmune response that contributes to the initiation of gingivitis.