We attempted to isolate the causative agent of Kuchijirosho of farmed tiger puffer using 8 established cell lines and a primary cell culture derived from the gonad of tiger. puffer (PFG). The inoculation of the brain or kidney filtrate prepared from diseased fish produced conspicuous CPE on PFG cells. However, no CPE was found in any of the cell lines used, i.e., BB, CHH-1, , CHSE-2l4, EPC, FHM, MCT, RTG-2 and SSE-30. Neither toxicity of the culture medium on PFG cells nor bacterial/mycoplasmal contamination was found, and the observed CPE was thus confirmed to be due to virus. The inoculation of the isolated virus to the tiger puffer resulted in high mortality and experimentally infected fish showed similar characteristic behavior and symptoms to those of naturally infected fish with Kuchijirosho. Furthermore, all experimental fish died after the inoculation of the viral fluid passed through a membrane filter with a pore size of 50 nm. Taken together the results of viral isolation, experimental infection and filtability test of isolated virus, it is concluded that Kuchijirosho is an infectious disease caused by a virus with a size less than 50nm in diameter.