Using enzyme-linked immunosorbent assay (ELISA), we measured anti-factor VIII immunoglobulin G (IgG), IgG4 and IgM in serum samples obtained from hemophilia A patients treated with a recombinant factor VIII (rFVIII) preparation (Kogenate). Twelve pre- and post-treatment serum samples from 3 previously untreated patients who developed an inhibitor (alloantibody) were evaluated. Both rFVIII and plasma-derived factor VIII (pdFVIII) were used as antigen. Serum samples were diluted 37.5-fold. For the measurement of IgM antibody, protein A Sephadex suspension was added to the patients' serum samples and the supernatant was assayed. Anti-human IgG, IgG4, and IgM monoclonal antibodies labelled with peroxidase were added and absorbance at 450nm was determined. The reactivity of the IgG antibody with rFVIII and pdFVIII was extremely low. Samples containing the IgG4 inhibitor with a neutralizing activity of approximately 7.5 Bethesda units (BU)/ml resulted in absorbance values of 0.451-0.551, thus demonstrating a considerably high degree of sensitivity. The correlation between the neutralizing activity and reactions of the IgG4 antibody with rFVIII and pdFVIII antigens was high, with correlation coefficients of 0.912 and 0.966, respectively. Furthermore, the correlation coefficient between the measured absorbance values for the antibody reacted with pdFVIII and rFVIII was 0.961. No correlation was found between the reactivity of IgM to rFVIII and pdFVIII.