Autoantibodies directed against cellular constituents rarely react with antigens localized in the Golgi apparatus and little information is available regarding these particular antibodies. Although thousands of samples have been examined for autoantibodies in our laboratory on a routine basis, only three human sera with anti-Golgi antibodies could be studied. Using pre-embedding immunoelectron microscopy we have demonstrated that these sera have antibodies reacting with antigens located in the Golgi apparatus. The reaction product was exclusively located on cisternal and vesicular Golgi membranes. No intraluminal staining was seen and some saccules were negative. No specificity for a peculiar tissue or cell line was noted, suggesting that the targets or these autoantibodies are evolutionarily conserved. The F(ab')2 fragments retained full binding capacity in indirect immunofluorescence experiments, confirming true antibody activity. When tested by immunoblotting, the three sera reacted with different antigens with relative molecular weights of respectively 230, 150 and 80 kDa. The antigens recognized by anti-Golgi antibodies in two of the three sera were insensitive to trypsin degradation. Together, these results suggest that a set of different autoantigens are recognized by sera from various patients.