DNA immunization induces CD8+ CTL responses by bone marrow-derived APCs, which are directly transfected with a plasmid DNA and/or acquire Ags from DNA-transfected non-APCs. To investigate the relative contribution of DNA-transfected APCs vs non-APCs to the initiation of CD8+ T cell responses, we used tissue-specific promoter-directed gene expression and adoptive transfer systems in gene gun DNA immunization. In this study, we demonstrated that non-APC-specific gene expressions induced significant CD8+ CTL and IFN-γ-producing cells and Ab responses, whereas APC-specific gene expressions led to moderate CTL and IFN-γ-producers, but no Ab responses. Interestingly, mice immunized with a non-APC-specific plasmid induced more rapid, vigorous, and prolonged proliferation of adoptively transferred Ag-specific CD8+ T cells than APC-specific plasmid-immunized mice. In addition, the in vivo proliferative responses elicited by a non-APC-specific plasmid administration were dependent on TAP, but were independent of CD4+ T cell help. Collectively, our results suggest that cross-priming, in which Ags expressed in non-APCs are taken up, processed, and presented by APCs, plays an important role in the initiation, magnitude, and maintenance of CD8+ T cell responses in gene gun DNA immunization.