We investigated embryo tissues targeted by replication competent adenovirus (Ad)-free recombinant Ad expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdH5) when injected into 18-day embryonated eggs. We also evaluated the effects of concurrent in ovo vaccination with the experimental AdH5 vaccine and commercially available Marek's disease virus (MDV) vaccine combinations Rispens/turkey herpesvirus (HVT) or HVT/SB-1. Computed tomography indicates that in ovo injection on day 18 of incubation places the solution in the amnion cavity, allantoic cavity, or both. Ad DNA was consistently detected in the chorioallantoic membranes as well as in the embryonic bursa of Fabricius, esophagus, and thymus 3 days postinoculation. H5 expression in these tissues also was detected by immunofluorescence assay. These results indicate possible swallowing of vaccine virus contained in the amnion. In contrast, vaccine localization in the allantoic fluid would have allowed bursal exposure through the cloaca. When the AdH5 vaccine was used in combination with MDV, chickens responding to the AdH5 vaccine had similar AI antibody levels compared with AdH5-only–vaccinated birds. However, combined vaccinated groups showed reduced vaccine coverage to AI, suggesting some level of interference. The combination of AdH5 with MDV Rispens/HVT affected the vaccine coverage to AI more severely. This result suggests that the replication rate of the more aggressive Rispens strain of serotype 1 may have interfered with the Ad-vectored vaccine. Increasing the Ad concentration produced similar AI antibody titers and AI vaccine coverage when applied alone or in combination with the HVT/SB-1 vaccine. Ad DNA was detected in hatched chickens 2 days after hatch but was undetectable on day 9 after hatch. MDV DNA was detected in feather follicles of all vaccinated birds at 12 days of age. Thus, Ad-vector vaccination does not interfere with the efficacy of MDV vaccination by using any of the commonly used vaccine strains.