The isolation of a marker antigen from rat liver and pancreas tissue, which reacts with antibodies in a subtype of autoimmune hepatitis by complement fixation test, enzyme-linked immunosorbent assay and Western blot, is described. The liver-pancreas antigen could be detected in tissue from different human or animal organs, but liver and pancreas yielded the highest activity. A highly specific antigen fraction was obtained by gel filtration and ion exchange chromatography with a 100,000 g supernatant from rat liver tissue, and this preparation was shown to be devoid of nuclear, mitochondrial and microsomal antigens and cytokeratin 8 and 18, as demonstrated by appropriate marker antibodies. These data and absorption studies with cell organelles indicate that liver-pancreas antigen is a cytosolic protein. By Western blotting, two major epitopes at molecular weights 52 kD and 48 kD could be visualized. Sera from 175 patients previously shown to have high complement-fixing activity to a nonpurified liver-pancreas antigen fraction were further analyzed. All were positive by enzyme-linked immunosorbent assay with the purified liver-pancreas antigen fractions, and 111 were also positive by Western blot. Eighty-six sera reacted with the 52-kD determinant, 33 with the 48-kD determinant and 2 with both determinants. In 117 of the 175 patients, antibody to liver-pancreas antigen was associated with other autoantibodies known to characterize subgroups of autoimmune hepatitis. Thus 19 patients had antibodies to nuclei and 96 to actin but none to liver-kidney microsomes, hereby suggesting that antibody to liverpancreas antigen may define another subgroup of autoimmune hepatitis. Only 38 (9%) of 425 patients with autoimmune hepatitis types 1 (lupoid hepatitis) and 2 (liver-kidney microsome 1 antibody–positive hepatitis) and 4 (3%) of 128 patients with chronic hepatitis B and C were positive for the antibody to liver-pancreas antigen as tested by enzyme-linked immunosorbent assay. None of the 196 patients with nonhepatic disorders had this antibody type. A classification of three different subgroups of autoimmune hepatitis is proposed: type 1a, lupoid hepatitis (antinuclear antibody positive with or without actin antibody); type 1b, only actin antibody positive; type 2, liver-kidney microsome 1 antibody positive; and type 3, liver-pancreas antigen antibody positive (with or without other autoantibodies). With this classification, group 3 comprises around 30%. (HEPATOLOGY 1993;18:1–9.