GAD65 is targeted by different patterns of autoantibodies [glutamic acid decarboxylase (GAD)-AAbs] in insulin-dependent diabetes mellitus (IDDM) and stiff-man syndrome (SMS). To study differentiation of the GAD-AAb pattern by immunohistochemistry, we examined the immunostaining of 15 monoclonal GAD antibodies (mc-GAD-Abs), which recognized different epitope regions of the antigen, on human pancreatic sections that were unfixed or fixed with different fixatives. By a competitive sandwich enzymelinked immunosorbent assay (ELISA), three binding patterns of mc-GAD-Abs were identified: 5 of 15 mc-GAD-Abs recognized a linear N-terminal epitope (p1), 5 of 15 were reactive with a conformational GAD65 epitope region (p2), and 5 of 15 were cross-reactive with GAD67 (p3). These patterns of mc-GAD-Abs were tested for islet cell binding by indirect immunofluorescence on pancreatic sections treated with either (1) Bouin's solution, (2) Zamboni's solution, or (3) phosphate-buffered formaldehyde for 0.5, 1, 2, and 18 h at 4°C. After fixation for up to 2 h no differentiation of immunoreactivity of patterns was observed using the three fixatives. mc-GAD-Abs recognizing conformational epitope regions (p2) revealed a marked reduced immunoreactivity on pancreatic sections fixed for 18 h with 4% formaldehyde, while mc-GAD-Abs reactive with linear epitopes (p1, p3) were detectable with strong binding. This fixation procedure was used to compare the immunoreactivity of GAD-AAb+or GAD-AAb−islet cell cytoplasmic antibody-positive (ICA+) sera of IDDM (n= 27) and SMS patients (n= 3). The three SMS sera were reactive with GAD on fixed islets but showed a reduced liter, whereas the majority of IDDM sera (22/27; 81.5%) were not detectable; 70.6% (12/17) of GAD-AAb+IDDM sera were not detectable on fixed islets. Furthermore, all 10 GAD-AAb−IDDM sera tested failed to react with fixed pancreas, which also suggested an alteration of non-GAD-ICA antigens. In conclusion, the fixation of human pancreatic sections with formaldehyde for 18 h allows the differentiation of GAD-AAbs recognizing linear and conformational epitope regions.