Infectious laryngotracheitis virus (ILTV), a member of the Alphaherpesvirinae, possesses several unique genes. One of them, UL0, encodes an abundantly expressed protein that accumulates in the nuclei of ILTV-infected cells. This study demonstrates that this protein is dispensable for in vitro virus replication and that UL0 deletion mutants exhibit only minor growth defects in cultured cells. The UL0 gene locus of ILTV was also used for insertion of foreign DNA sequences encoding enhanced GFP or haemagglutinin (HA), subtype H7, of a highly pathogenic avian influenza virus under the control of the human cytomegalovirus immediate-early gene promoter. Expression of foreign proteins was shown by (immuno)fluorescence tests and Western blot analyses. After experimental infection of chickens, UL0 deletion mutants proved to be attenuated when compared to both parental wild-type ILTV and an UL0 rescue mutant. Nevertheless, all animals immunized with UL0-negative ILTV were protected from clinical disease after subsequent infection with virulent ILTV. Furthermore, all animals immunized with HA-expressing ILTV survived a lethal challenge with H7 subtype avian influenza virus with minimal clinical signs. Thus, an UL0-negative and HA-expressing ILTV recombinant may be used as a bivalent live virus vaccine against ILT and fowl plague. Unlike inactivated influenza virus vaccines, HA-expressing ILTV recombinants should be suitable for mass application and would also permit serological discrimination between vaccinated and virus-infected animals in the field.