Bovine herpesviruses type 1 and 5 (BoHV-1 and BoHV-5) are closely related yet differ markedly in their neuropathogenic potential. BoHV-1 isolates have been associated with respiratory and genital disease whereas BoHV-5 has been consistently isolated from neurological infection. We report the characterization of five Brazilian BoHV-1 isolates associated with neurological disease, an unusual finding. All five viruses were isolated from the brain of cattle presenting neurological disease, yet prominent histological encephalitis was not observed in three cases. The isolated viruses were identified as BoHV-1 by a glycoprotein C gene-based PCR able to differentiate BoHV-1 from BoHV-5. The identity of the isolates was confirmed by nucleotide sequencing of the amplicons and by restriction analysis of PCR products from another gC region. Monoclonal antibody binding and cross-neutralization assays with BoHV-1 and BoHV-5 antisera showed a typical BoHV-1 antigenic profile. Lastly, inoculation of rabbits with these five BoHV-1 isolates did not result in neurological disease, contrasting with fatal meningoencephalitis produced by BoHV-5. Thus, the involvement of BoHV-1 in neurological disease of cattle is more frequent than previously reported, indicating the need for fast and precise means of differentiating it from BoHV-5. Likewise, the potential role of BoHV-1 in neurological infection in cattle should be further investigated.