Technical Abstract: Recombinant replication-defective human adenovirus type 5 (Ad5) vaccines containing capsid-coding regions from foot-and-mouth disease virus (FMDV) have been demonstrated to induce effective immune responses and provide homologous protective immunity against FMDV in cattle. However, basic mechanisms of vaccine function including virus tropism, transgene expression, and antigen presentation, remain incompletely understood. The current study characterized dynamics of adenovirus infection in cell lines and early vector-host interactions in cattle immunized with two Ad5 constructs (Ad5- FMDV-A24 and Ad5-Luciferase). Ad5 dissemination was described utilizing novel rPCR, rRT-PCR, luminometry, and immunomicroscopy techniques. In vitro infection of human and bovine cells with both Ad5 vectors resulted in dose-dependent detection of vector DNA, mRNA transcripts, and transgene-encoded antigens. Subsequent to intramuscular inoculation of cattle, Ad5 and transgene products were detected at the injection sites of all animals at all time-points examined (6-48 hpi). Microscopically, injection sites were characterized by marked infiltrates of interstitium consisting of predominantly large mononuclear cells. Immunomicroscopy indicated these cells infrequently contained adenovirus and/or transgenic proteins and were phenotypically consistent with macrophages and dendritic cells. Vector DNA and mature mRNA transcripts were first detected at the local lymph nodes as early as 6 hpi and systemically at 24hpi. These results provide novel insights for understanding Ad5-mediated immunity against FMDV using novel techniques that will contribute to ongoing efforts for the improvement of future vaccine platforms.