T-cell receptor-mediated T-cell activation requires cosimulation signal, which can be provided by B7-1 molecule. Our previous study demonstrated that the coexpression of a covalent peptide/major histocompatibility complex class II molecule complex and costimulatory molecule B7-1 by recombinant adenovirus leads to synergy in peptide-specific T-cell activation. However, the viral antigen-specific T-cell activation is not enhanced by B7-1 expressed by the adenovirus. To verify the differential T cell activation by B7-1 and investigate its underlying mechanisms, we constructed an adenovirus coexpressing a covalent complex of hen egg lysozyme peptide/I-Ak (HEL46-61/I-Ak) and B7-1 in the present study. In vivo studies revealed that HEL46-61-specific T-cell response, but not viral antigen-specific T-cell response, was enhanced by B7-1 expression mediated by the adenovirus, suggesting that exogenous B7-1 expression may regulate T-cell response to these two different antigens through distinct mechanisms. Furthermore, our results revealed that antigen-presenting cells were not susceptible to adenovirus infection in vivo. Based on these findings, the possible mechanism of differential B7-1 costimulation on peptide-specific and viral antigen-specific T-cell activation is discussed.