|Protective immunity to avian influenza (AI) virus can be elicited in chickens by in ovo or intramuscular (IM) vaccination with RCA-free adenovirus vector encoding AI virus H5 (AdH5). We evaluated persistence of specific antibody after in ovo vaccination, transfer of antibody to progeny chickens, and possible interference of maternal antibody with in ovo and mucosal immunization. One flock of breeder birds was vaccinated in ovo-only, another flock was vaccinated in ovo and boosted IM on week 16 of age, and a third group was the unvaccinated control. AdH5 in ovo vaccination elicited anti-H5 hemagglutination inhibition (HI) antibodies in breeder hens for up to 34 weeks of age. Intramuscular revaccination of in ovo vaccinated hens increased HI antibodies significantly. Breeder hens vaccinated in ovo with AdH5 effectively transferred anti-H5 maternal antibodies to progeny chickens. Maternally derived anti-H5 antibodies (MDA) from breeders vaccinated in ovo and boosted with AdH5 interfered with active immunization in progeny chickens.