A thymidine kinase-deficient (TK('-)) mutant of pseudorabies virus (PrV) was isolated from the virulent Indiana-Funkhauser strain, by selection for resistance to bromodeoxyuridine and arabinofuranosylthymine. The TK('-) mutant did not revert to TK('+), and was avirulent for mice and young swine. The TK('-) mutant grew as well in cell culture as the TK('+) parental strain, and was not temperature sensitive at 38.5 C. The TK('-) mutant was tested as a vaccine in mice and young swine. Vaccinated mice were protected from mortality, and vaccinated swine were protected from mortality and severe morbidity after challenge with lethal doses of a virulent PrV field strain. Two PrV field isolates and 3 commercial modified live virus (MLV) vaccine strains (PR-Vac, Norden Laboratories, Inc; PseudoVax, Pitman-Moore, Inc; Pseudorabies Vaccine, Bioceutic Laboratories, Inc.) were compared for mouse virulence and for TK activity. All three MLV strains produced fatal infections in mice, but were reduced in virulence 2- to 10-fold relative to the field strains. The 3 MLV strains and 2 field isolates were TK('+), and Km values for TK from all strains ranged from 2.9 to 3.9 (mu)m. Restriction enzyme (RE) analysis of viral DNA revealed that 2 of the MLV strains were closely related, and all 3 MLV strains contained a major deletion in the Pst I A fragment. RE analysis also revealed small increases in molecular weight of fragments mapping to the repeat sequences of the TK('-) mutant when compared to the TK('+) parental strain.