Investigation into the genetic stability of a replication-competent human adenovirus rabies glycoprotein recombinant (ONRAB) developed for use as an oral vaccine for wildlife rabies prevention is of major importance due to the vaccine's intended placement in the environment. Using a collection of murine monoclonal antibodies directed to six distinct antigenic sites on the rabies glycoprotein, preservation of all main immunogenic epitopes of the protein after virus growth in vitro was established. A competition experiment which involved the in vitro passaging of a mixture of ONRAB and wild-type human adenovirus type 5 demonstrated that the two viruses do not exhibit noticeably different fitness levels in this environment. Nucleotide sequencing of the expression cassette of multiple viral clones recovered after 20 serial passages in cell culture and 5 serial passages in cotton rats (Sigmodon hispidus), a species susceptible to human adenovirus infection, indicated no changes in comparison to the original virus. These trials demonstrated the stability of the insert gene of ONRAB during in vivo and in vitro passaging.