The objective of this study was to determine if a replication defective recombinant adenovirus expressing rabies virus glycoprotein (Adrab.gp) given through a non-invasive vaccination route (by topical application) onto the skin (NIVS) could elicit an immune response and/or protection against rabies. Groups of mice were immunized by NIVS with various doses of Adrab.gp. For comparison, groups of mice were immunized intramuscularly, subcutaneously, or intradermally with Adrab.gp. Mice received two booster immunizations at 1 and 2 months after the first immunization. Virus neutralizing antibody (VNA) titers were measured at day 21 after the first and second immunizations and at day 14 after the third immunization. Fifty percent of the mice immunized by NIVS with 2 x 10(7) and 2 x 10(8)pfu Adrab.gp vaccine developed VNA, whereas none of the control mice or the mice immunized by NIVS with the lowest dose (2 x 10(6)pfu) of Adrab.gp virus developed VNA. However, this low dose induced high titers of VNA in mice immunized by parenteral routes. Two weeks after the last immunization, all the mice were challenged with a lethal dose of rabies virus. More than 70% of the animals immunized by NIVS with > or = 2 x 10(7)pfu Adrab.gp virus survived the challenge, whereas all the mice in the negative control group and the group immunized by NIVS with the lowest dose of Adrab.gp succumbed to rabies. Taken together, the results suggest that NIVS with Adrab.gp can induce VNA production and protection against lethal challenge with rabies virus in mice.