Pseudorabies virus (PrV) is the causative agent of Aujeszky's disease which results in significant losses in pig husbandry. Recently we identified the gene encoding the deoxyuridine-triphosphatase (dUTPase) of PrV as the homolog of the UL50 gene of herpes simplex virus type 1. The PrV UL50 gene product was characterized and a UL50 negative PrV mutant (PrV UL50-) was generated by insertion of a lacZ expression cassette into the UL50 open reading frame (Jöns and Mettenleiter, J. Virol. 70, 1242-1245). Here we show that replication of PrV UL50- in cell culture was only slightly impaired as compared to wild-type PrV strain Ka. After intranasal infection of young pigs PrV UL50- proved to be substantially attenuated, whereas severe clinical signs and death occurred after infection with wild-type PrV. Challenge infection with the highly virulent NIA-3 strain of PrV showed that prior infection with PrV UL50- conferred protection against Aujeszky's disease. Innocuity and efficacy make UL50-negative PrV an attractive candidate for a live PrV vaccine.