The latency-related (LR) gene of bovine herpesvirus 1 (BHV-1) is abundantly expressed during latency. A mutant BHV-1 strain that contains three stop codons at the 5' terminus of the LR gene (LR mutant) does not reactivate from latency. This study demonstrates that the LR mutant does not express open reading frame 2 or an adjacent reading frame that lacks an initiating ATG (reading frame C). Since the LR mutant and wild-type BHV-1 express similar levels of LR RNA, we conclude that LR protein expression plays an important role in regulating the latency reactivation cycle in cattle.