Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4+ Th2-type, cytokine-mediatcd mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4+ T cell proliferative responses and OVA-induced IFN-γ and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CDllb+CDllc+ DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CDllb+CDllc+ DCs that migrate to effector sites for subsequent CD4+ Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.