Analysis of the live attenuated pseudorabies virus (PrV) vaccine strain Bartha indicated location of a major determinant for PrV neurovirulence within the genomic BamHI fragment 4 (B. Lomniczi et al., 1984, J. Virol. 52, 198-205). To more precisely localize the defect, marker rescue experiments were performed using cloned subfragments of BamHI-4. Rescuants were analyzed after intracerebral infection for their virulence in chicken, as well as after intranasal infection for virulence in pigs. We show that the defect associated with attenuation in strain Bartha is located in a 3.8-kb subfragment of BamHI-4 which encompasses the PrV UL20 and UL21 genes and a putative origin of replication (B. Klupp, H. Kern, and T. C. Mettenleiter, 1992, Virology 191, 900-908). Sequence analysis of this region of the strain Bartha genome and comparison with the corresponding region in wild-type PrV strain Ka revealed the presence of eight point mutations. Four nucleotide exchanges reside within the UL21 gene with three of them leading to amino acid substitutions; one is located in the intergenic region between the UL20 and UL21 genes and three are localized downstream from the UL21 gene. Neither the UL20 gene nor the putative origin sequence was affected. Insertional inactivation of the UL21 gene in wild-type PrV strain Ka led to a marked attenuation of the virus for pigs infected by the intranasal route. In summary, our data show that the PrV UL21 gene is a major determinant of PrV virulence and that point mutations affecting the UL21 gene of live vaccine strain Bartha contribute to its attenuated phenotype.