The occurrence of antibodies to interferon-alpha 2a (anti-IFN-alpha 2a) to recombinant human IFN-alpha 2a was examined in chronic liver disease by a sensitive enzyme-linked immunosorbent assay (ELISA). Naturally occurring IgG and/or IgM anti-IFN-alpha 2a were found in one of 12 cases of chronic persistent hepatitis, four of 18 cases of chronic active hepatitis (CAH), two of 12 cases of liver cirrhosis, six of seven cases of primary biliary cirrhosis, nine of 11 cases of auto-immune CAH and none of 21 normal control subjects. Fifteen patients with viral CAH were treated with recombinant IFN-alpha 2a. Two of them were positive prior to receipt of IFN-alpha 2a and their titres increased after the therapy. Two patients became positive for anti-IFN-alpha 2a after the therapy. Absorption experiments revealed that anti-IFN-alpha 2a cross-reacted with native human leucocyte IFN-alpha and recombinant IFN-alpha 2b but not with recombinant IFN-beta and -gamma. The immunoblotting experiment confirmed the binding of antibodies to IFN. The results of anti-IFN-alpha 2a obtained by antiviral, cytopathic effect assay were in good agreement with those of IgG anti-IFN-alpha 2a, but not with those of IgM antibodies obtained by the ELISA. The ELISA described in the present study is a simple, sensitive and quantitative assay for anti-IFN-alpha 2a. It should be useful in assessing sub-specificities of anti-IFN and provide valuable information to predict the effect of IFN therapy and to elucidate the immunological abnormality in liver disease.