The difference between the attenuated live vaccine strain 758-43 and its parent virulent strain 758 was investigated genetically. These viruses were propagated in Madin–Darby bovine kidney cells, and viral DNA was obtained from the culture supernatants of the infected cells. Based on a previous report, a large deleted region would seem to exist in the Hind III J fragment located between nucleotide numbers 2439 and 11,270. Three pairs of primers were designed based on the complete BHV-1 DNA sequence. With one pair of primers used, the PCR products derived from strains 758 and LA resulted in fragment sizes of 1850bp, whereas that from the vaccine strain was smaller than those from the virulent strains. The attenuated live vaccine strain, 758-43, lacked 652bp in the PCR product region, accounting for approximately 84% of the coding region of the UL49 homolog gene of BHV-1. The present results provide a new and important information to distinguish the vaccine strain 758-43 clearly from wild-type BHV-1 isolates in Japan. The UL49 homolog gene seems to participate in pathogenicity in herpesvirus infections.