The use of factor VIII prepared genetically engineered cell lines (rFVIII) may avoid some of the problems inherently associated with administering plasma-derived factor VIII (pdFVIII) concentrates to hemophilia A patients. Although rFVIII may represent an improvement over traditional therapeutics, the chance exists that protein production in cell culture may result in the presence of novel epitopes that may enhance the formation of inhibitor antibodies capable of neutralizing either rFVIII or pdFVIII. To assess the differences between rFVIII and its plasma-derived homologue, a rabbit immunogenicity model system was developed. Antibodies raised to rFVIII in rabbits were tested for the presence of antibodies capable of binding rFVIII but not pdFVIII, the presence of which would suggest that novel epitope(s) were present. This analysis was performed using a competitive enzyme-linked immunosorbent assay, as well as immunoadsorption. For either technique, rFVIII-specific antibodies were not detected, indicating that differences between rFVIII and pdFVIII were not found. When a rFVIII B-region deletion mutant was similarly tested, antibodies specific for this protein were found. These specific antibodies appeared to bind in the vicinity of the deletion site and their binding was not affected by carbohydrate removal. These results indicate that the rabbit immunogenicity model system is sensitive to alterations in the factor VIII molecule and suggest that full-length rFVIII will not be any more immunogenic in human patients than pdFVIII.