Envelope glycoprotein gp50 of pseudorabies virus (PRV) is essential for virus entry, but is not required for subsequent steps in the viral replication cycle. Phenotypically-complemented gp50 null mutants can infect cells and can spread, both in vitro and in vivo, by direct cell-to-cell transmission. However, progeny virions released by the infected cells are non-infectious because they lack gp50. Therefore, these viruses cannot be transmitted from infected animals to contact animals. These properties could make PRV gp50 null mutants attractive candidates as safe non-transmissible live vaccines. To establish whether phenotypically-complemented PRV gp50 null mutants and gp50 + gp63 double mutants could be used as live vaccines against Aujeszky's disease, the virulence and immunogenicity of these mutants were tested in pigs. Our results show that a gp50 null mutant has a greatly reduced virulence for pigs, and that pigs immunized with such a mutant were protected from clinical signs of Aujeszky's disease after a challenge inoculation with the virulent wild-type PRV strain NIA-3. PRV gp50 + gp63 deletion mutants proved to be non-virulent for pigs and were somewhat less immunogenic, since immunized animals showed some fever and growth retardation after challenge inoculation. Replication of wild-type challenge virus was significantly reduced, but could not completely be prevented, in pigs immunized with a gp50 null mutant, and was reduced less in pigs immunized with a gp50 + gp63 deletion mutant. Furthermore, infectious virus could not be recovered from oropharyngeal fluid or tissues from pigs inoculated with a gp50 null mutant or a gp50 + gp63 deletion mutant.(ABSTRACT TRUNCATED AT 250 WORDS)