A series of monoclonal antibodies was prepared against purified adenovirus type 2 fiber. These antibodies were produced by both classical techniques and by electrofusion. Fifty antibodies were obtained, and five were selected which had the following characteristics: (i) they were able to reduce adenovirus 2 infection of Hep-2 cells; (ii) they had different light-chain structures and (iii) they did not react with denatured fiber. The five antibodies (AdF18, AdF38, AdF41, AdF2D3, AdF3Bl) recognized between them three periodate-insensitive epitopes on the fiber. AdF18 bound to both adenovirus type 2 and type 5; all the others were specific to type 2. Polyclonal anti-idiotype antibodies to these antibodies were produced in rabbits and were purified extensively on immunoaffinity columns. Anti-idiotypes to AdF38 and AdF41 were able to inhibit virus infectivity, and anti-AdF41 competed with fiber for binding to the surface of Hep-2 cells.