Adenoviral transfer of human apo A-I in Balb/c mice induces a strong humoral immune response against the transgene product when expression is driven from the ubiquitously active CMV promoter but induces no immune response when driven by the hepatocyte-specific 256-base pair apo A-I promoter. Here the hypothesis was tested, which is that the humoral immune response against the circulating transgene product correlates with its expression in antigen-presenting cells. No humoral immune response was observed after adenoviral transfer of vectors with human apo A-I expression driven by the hepatocyte-specific apo C-II or 1.5-kilobase (kb) human alpha(1)-antitrypsin promoter, but antibodies were induced after transfer with vectors driven by the ubiquitously active U1b promoter and the murine MHCII E beta promoter. A strict correlation was observed between antigen expression in the spleen and the occurrence of an immune response. Coinjection of the 1.5-kb human alpha(1)-antitrypsin and the murine MHCII E beta promoter-driven vectors resulted in a very short-lived humoral immune response against human apo A-I, suggesting that the time course of human apo A-I expression is a critical determinant of the development of tolerance for human apo A-I. High titers of antibodies against human apo A-I after subcutaneous gene transfer with the MHCII E beta promoter-driven vector underscore the potential of this promoter for vaccination purposes. In conclusion, humoral immune response in mice against a circulating antigen induced by adenoviral transfer is strictly dependent on expression in antigen-presenting cells.