A Japanese strain (Yamaguchi) of bovine ephemeral fever virus, propagated in BHK21-WI2 cells of hamster kidney origin, was used. Adult rats inoculated intracardially or intracerebrally remained healthy, but produced neutralizing antibody, suggesting actual viral growth in the animals. However, no clinical and serological evidence for infection with the virus was obtained in adult sheep, goats, swine and rabbits inoculated intravenously.One-day-old guinea pigs inoculated intracerebrally and adult guinea pigs inoculated intracardially remained healthy and produced no neutralizing antibody. The virus multiplied not only in BHK21-WI2 cell culture but also in cultures of stable line hamster cells (HmLu-1 and HmT), primary hamster kidney and embryo cells, long-term cultured bovine embryo cells (BEK1), primary calf kidney and embryo cells, and primary suckling rat kidney and embryo cells. However, no evidence for cytopathogenesis and viral growth was obtained in primary cultures of bovine leukocytes, bovine bone marrow, thymus, thyroid and adrenal cells, guinea pig, mouse and chick kidney cells, and mouse and chick embryo cells, and in culture of African green monkey kidney cells (Vero). The virus multiplied best and induced marked cytopathogenesis in BHK21-WI2 cells; lower temperatures such as 34 C or 30 C appeared preferable to obtain constantly better viral yields, although the virus grew more slowly. Similar effects of the incubation temperatures were also obvious in the other susceptible cell cultures, in which the cytopathogenesis was less marked than in BHK21-WI2 cells or absent, roughly coinciding with decreasing grades of viral growth.