Dendritic cells (DCs) are highly specialised antigen-presenting cells (APCs) that are essential for the initiation and modulation of T cell-mediated immune responses. In order to induce effective CTL responses against most infections and tumours, DCs must prime both CD4⁺ and CD8⁺ antigen-specific T cells. It is, therefore, important in vaccine design to produce antigen-delivery systems that lead to the simultaneous presentation of multiple histocompatibility complex (MHC) class I- and class II-restricted antigenic peptides by DCs. In this study, the infection of immature mouse bone marrow-derived DCs (BMDCs) with recombinant adenovirus (rAd) vectors led to a marked upregulation of surface costimulatory molecules, IL-12 p40 production and capacity to stimulate both allogeneic and antigen-specific T cells. Furthermore, infection of immature and mature BMDCs with a rAd encoding chicken ovalbumin (OVA) led to presentation of the antigen to TCR-transgenic OVA-specific CD4⁺ and CD8⁺ T cells. In addition, the activation state of responding CD8⁺ T cells was further amplified if they recognised antigen on rAd-transduced BMDCs in the presence of antigen-specific CD4⁺ T cells. The results suggest that rAd-encoded OVA protein is secreted by BMDCs, taken up by endocytosis and presented in association with MHC class II molecules for activation of OVA-specific CD4⁺ T cells. Consequently, rAd-transduced immature BMDCs become better stimulators of antigen-specific CD4⁺ T cells than rAd-infected mature BMDCs. Taken together, these data have important implications for vaccine design, and suggest that infection of immature DCs with rAd encoding MHC class I and class II-restricted T cell epitopes could be an efficient means of inducing effective immune responses.