Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF. IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (iv) injection of vector. For example, iv injection of an adenoviral (Ad) vector results in sustained expression of hF. IX in normal or hemophilic C57BL/6 mice, while anti-hF. IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF. IX with Ad-hF. IX vector via peripheral vein. All mice expressed hF. IX antigen without detectable anti-hF. IX, even when challenged with hF. IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF. IX titers diminished to undetectable levels after iv administration of Ad-hF. IX. Lymphocytes from mice that had received Ad-hF. IX iv failed to proliferate when stimulated with hF. IX in vitro after the animals had been repeatedly challenged with hF. IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF. IX in C57BL/6 mice after iv injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF. IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.