We generated both replication-incompetent (HAd5-gD-E1 and HAd5-tgD-E1) and replication-competent (HAd5-gD-E3 and HAd5-tgD-E3) human adenovirus type 5 (HAd5) recombinants expressing the full (gD) or truncated form (tgD) of the glycoprotein gD gene of bovine herpevirus type 1 (BHV-1). Recombinant gD and tgD expressed by HAd5-gD-E1 and HAd5-gD-E3 and by HAd5-tgD-E1 and HAd5-tgD-E3, respectively, were recognized by gD-specific monoclonal antibodies (MAbs) directed against linear and conformational epitopes, suggesting that antigenicity of recombinant gD and tgD was similar to that of the native gD expressed in BHV-1 infected cells. In HAd5-gD-E1- or HAd5-gD-E3-inoculated cotton rats there was a strong gD- and HAd5-specific IgG and IgA antibody response. The immune response was significantly lower in animals similarly immunized with HAd5-tgD-E1 or HAd5-tgD-E3, indicating that live adenovirus vaccine vectors may be better suited to the full-length form of glycoprotein gD than its truncated form. After a BHV-1 challenge, no infectious BHV-1 virions were isolated from the trachea of cotton rats previously immunized with HAd5-gD-E1 or HAd5-gD-E3. These results suggest that adenovirus E1 insertion (replication-incompetent) and E3 insertion (replication-competent) vectors have excellent potential for use in developing live recombinant virus vaccines and provide evidence that the cotton rat model can be used in BHV-1 vaccination-challenge trials.