Bovine herpesvirus type 1 (BHV-1) causes respiratory and genital diseases in cattle, whereas the closely related BHV-5 can induce severe meningoencephalitis in calves. To characterize BHV-5 glycoprotein C (gC5) within the backbone of BHV-1, three consecutive recombinant viruses were constructed: A deletion mutant (rBHV-1delta gC blue) with gC replaced by the lacZ gene, an exchange mutant (rBHV-1gC5) with the lacZ of BHV-1delta gC blue exchanged by gC5, and a rescue mutant (rescue BHV-1) from rBHV-1gC5 with an additional XbaI site in gC1. The recombinant and wildtype viruses were characterized on MDBK cells. Although no significant differences were observed in growth behaviour and entry kinetics, rBHV-1gC5 showed a distinct phenotype in a heparin blocking assay. The gC5 was able to transfer the heparin binding phenotype of BHV-5 to BHV-1. This indicates that gC1 and gC5 differ in their receptor binding qualities, which might modulate the ability of the viruses to spread within the central nervous system.