Microsomal antigen autoantibodies are typical of type 2 autoimmune hepatitis, and a strong association with chronic hepatitis C virus (HCV) infection has been reported in certain geographical areas. These autoantibodies have been denominated LKM-1 to differentiate them from those associated with thienylic acid-induced hepatitis (LKM-2) and from those seen in patients with chronic delta hepatitis (LKM-3). To investigate the antigenic specificity of autoantibodies associated with chronic hepatitis C and delta, we analyzed 52 LKM-1 positive serum samples from patients with chronic hepatitis C and 17 LKM-3 positive serum samples from patients with chronic delta hepatitis by indirect immunofluorescence and Western blotting (immunoblotting). Reactivity of subjects with chronic hepatitis C was heterogeneous: only 5 out of 52 LKM-1 positive patients, tested by Western blot, recognized a single protein of 50 kD, previously identified by Manns et al. with an immunogenic epitope of cytochrome P450IID6. Thirteen of the 52 patients also reacted with a 70 kD microsomal protein, and 12 out of 52 reacted only with a 59 kD protein. Twenty-two sera, notwithstanding the high titer in immunofluorescence, did not evidence any reactivity when tested by Western blot. The same sera tested positive in LKM-1 ELISA when solubilized human microsomal proteins were used. Fourteen out of 17 LKM-3 positive sera from patients with chronic hepatitis delta recognized a 55 kD microsomal protein in Western blot; three sera, HCV and HIV positive, did not react with any protein by Western blot. None of these sera was positive in ELISA LKM-1.(ABSTRACT TRUNCATED AT 250 WORDS)