Clinical, pathological and virological examinations were carried out on naturally occurring cases of canine herpesvirus (CHV) infection. The first clinical sign observed was diarrhea, followed by anorexia, vomiting, serous nasal discharge and dyspnea. Some puppies had continual crying, abdominal pain and incoordination. The charactetistit lesions of CHV infection consisted of disseminated focal necrosis, hemorrhages and the appearance of intranuclear and cytoplasmic inclusion bodies in many organs and tissues. Furthermore, characteristic herpesvirus particles were detected by electron microscopy in the typical intranuclear and cytoplasmic inclusions found in occasional hepatic cells. Herpesvirus was isolated from the various organs of eight affected puppies. The isolate (GCH-1), which was propagated in DK cells, was similar to other herpesviruses in physicochemical and morphological characteristics, and was immunologically related to the known CHV strains. The GCH-1 strain, following inoculation into susceptible puppies, produced the typical clinical signs and lesions that were found in the natural case. In a serological survey, neutralizing antibodies against the virus were not found in 130 sera obtained from local mongrel dogs in the Aichi and Gifu prefectures. This paper reports the first naturally-occurring outbreak of CHV infection of neonatal puppies in Japan.