An experimental transmission study aimed at fulfilling Koch's postulates for a herpesvirus-associated stomatitis-rhinitis in Mediterranean tortoises is presented. Clinical, pathologic, serologic, and molecular studies were performed linking tortoise herpesvirus with the pathogenesis of stomatitis-rhinitis. Four adult Greek tortoises received either intranasally or intramuscularly two tortoise herpesvirus isolates by primary experimental infection and secondary challenge 11 months later. After the primary experimental infection and the secondary challenge, clinical signs of illness developed, which included conjunctivitis, diphtheritic oral plaques, and oral discharge. At 4 weeks after the secondary challenge, all tortoises were humanely euthanatized and evaluated. Although neutralizing antibodies developed after the primary experimental infection, they apparently did not prevent the later development of recurrent clinical signs. Polymerase chain reaction (PCR) and reverse transcription-PCR analyses allowed sensitive characterization of the systemic distribution of the herpesvirus DNA sequences and their presence in the cranial nerves and brains of the infected tortoises. Despite the failure to recover the herpesviruses used in the transmission study, the findings support the premise that tortoise herpes-virus is a primary pathogen of Greek tortoises.