Human parainfluenza virus type 3 fusion (F) and hemagglutinin-neuraminidase (HN) cDNA sequences were inserted into the E3 region of the adenovirus type 5 genome. Cells infected with recombinant adenoviruses containing HPIV3 F (AdF) and HN (AdHN) sequences were shown to express HPIV3 F and HN proteins that were functional and immunogenic. The HN protein produced following AdHN infection was glycosylated, expressed on the surface of infected cells and exhibited both hemagglutinin and neuraminidase activities. AdF infection led to the synthesis of both the HPIV3 F0 precursor and its proteolytic cleavage product, F1. F proteins produced by AdF were glycosylated and expressed on the infected cell surface. Syncytium formation was observed in HeLa T4 cell monolayers upon coinfection with AdF and AdHN. The F and HN proteins expressed by recombinant adenoviruses were recognized by HPIV3 F- and HN-specific monoclonal antibodies. Mice injected intraperitoneally with AdF or AdHN produced antibodies that immunoprecipitated the appropriate HPIV3 glycoproteins and sera from immunized mice effectively neutralized HPIV3 virions. These results support future work using recombinant adenoviruses to study the immune response to individual HPIV3 glycoproteins as well as in protection studies using animal models.