Bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 5(BoHV-5) are two closely related alphaherpesviruses thatinfect cattle. Both viruses are neurotropic but only BoHV-5 significantly replicates in the central nervous system andinduces neurological disease. We previously isolated 2interspecific recombinant viruses (R1 and R2) betweenBoHV-1 and BoHV-5 [1]. We report here on the precisecharacterization of the genetic backgrounds and on thevirulence assessment of these two BoHV-1/5 recom-binants in their natural host.An exhaustive PCR/sequencing approach led us to iden-tify the precise location of the two cross-over (CO) pointswhere recombination events occurred between the paren-tal strains. The first CO occurred in a 44 base pairs (bp)sequence of homology at the 3' end of the UL28 encodinggenes of BoHV-1 and BoHV-5. The resulting recombinant(R1) inherited 37% of the BoHV-1 genome. The rest of itsgenome (63%) is homologous to BoHV-5 containing thefinal region of the unique long (UL) region (from UL27 toUL0.5), the internal and terminal repeats (IR and TR) andthe unique short region (US). The second CO occurred ina 39 bp sequence of homology within the UL43 gene. Theresulting recombinant (R2) is mainly composed of BoHV-1 sequences (86%). It is homologous to BoHV-1 in a largeportion of the UL region (from UL43 to UL0.5), in the IR/TR and in the US region. BoHV-5 sequences account for14% of its genome content.In order to study the