Regional lymph nodes (RLNs) possess important immune functions and represent a major pathway of metastasis for solid tumors. Given these facts, the ability to transfer exogenous genes to the RLNs with the goal of manipulating the local immunological milieu would be desirable. On the basis of the hypothesis that a significant proportion of adenovirus (Ad) gene transfer vectors traffic through the lymphatics, E1-E3-Ad vectors were injected into the hind footpad of C3H/He mice and the RLNs assessed for vector trafficking and transgene expression. A low dose (109 particles) of an Ad vector encoding the firefly luciferase gene (AdCMV.Luc) resulted in luciferase expression only in the injection site and RLNs, with no detectable systemic (liver, spleen, lung) expression. At a higher dose (10¹¹ particles), some expression could be detected systemically in addition to the RLNs, but at levels in liver 14-fold less than in the RLNs. Transgene expression in the RLNs was transient, peaking at 1 day, decreasing markedly by 7 days. At high doses (10¹¹ particles), interruption of draining lymphatics decreased the amount of systemic dissemination 22-fold, suggesting that a large proportion of the vector trafficks through the lymphatics before reaching the systemic circulation. Administration of a vector encoding the jellyfish green fluorescent protein gene (AdCMV.GFP, 10¹¹ particles) showed that transgene expression in the RLNs was primarily in the cortical area. After footpad injection of a fluorescent-labeled Ad vector (Cy3-AdCMV.Null), fluorescent virions were visualized in the draining lymph. Regional lymph collected from animals injected in the footpad with AdCMV.Luc (10¹¹ particles) contained functional vector. Augmentation of local immune function in the RLNs was achieved by footpad administration of an Ad vector encoding murine IL-12, resulting in high mIL-12 and IFN- gamma levels in the regional, but not distant, nodes. These data demonstrate that expression of exogenous genes in RLNs is easily accomplished with Ad vectors, Ad vector dissemination occurs primarily via the lymphatics after footpad administration in mice, and basic immune functions in the RLNs can be manipulated by Ad-mediated gene transfer in vivo.