Kuchijirosho is a fatal disease of commercially cultured fugu, Takifugu rubripes. The transmissible nature of kuchijirosho strongly suggests that an infectious pathogen is the causative agent. Because it is filtrable, the agent is thought to be a virus; however, it has not yet been identified. The lack of a permissive cell line for the putative kuchijirosho-causing agent (KCA) has hindered research on the identification of this pathogen. We inoculated brain extract prepared from kuchijirosho-affected fugu onto an established fugu cell line, fugu eye, and observed that cytopathic effect appeared 7 days after inoculation. Injection of the culture medium of infected fugu eye cells into fugu resulted in the onset of kuchijirosho, indicating that fugu eye cells are able to proliferate KCA. An infectious fraction separated by sodium iottalamate density gradient centrifugation showed a density of 1.15 g mL(-1) equivalent to that of KCA derived from affected fugu brain. To determine whether the genome of KCA is RNA or DNA based, nucleotide synthesis inhibitors were applied to inoculated fugu eye cell line to influence the production of KCA. 5-Fluorouracil but not IUdR showed a concentration-dependent inhibition of KCA yield. These results suggest that KCA is an RNA virus.