A deletion was introduced into the thymidine kinase (TK) gene of the BHV1 strain Lam and, or, the complete coding region of the glycoprotein E (gE) gene was deleted to reduce virulence and to make serological differentiation possible. The virulence and immunogenicity of these three BHV1 mutants (TK-, gE- and TK-/gE) were studied in specific-pathogen-free calves. Although inactivation of TK strongly reduced the virulence of the Lam strain, deletion of the gE gene alone sufficed to yield complete attenuation of the Lam strain for seven-week-old calves. The three mutants induced protective immunity against disease after challenge with a virulent BHV1 strain. The reduction of virus shedding after challenge was related to the virulence of the various strains. The immunogenicity of the mutants was also evidenced by the reduction of challenge virus shedding after dexamethasone treatment. None of the mutant viruses could be isolated after dexamethasone treatment. The results demonstrate that the gE- and TK-/gE- mutants are good candidates for incorporation in a BHV1 marker vaccine.