Heterobothrium okamotoi, a monogenean gill parasite, shows high host specificity for tiger puffer, Takifugu rubripes. In the present study, in vivo and in vitro experimental infections were conducted using various fish species, including T. rubripes, to understand the mechanisms of specificity. In in vivo experiments, T. rubripes, grass puffer, Takifugu niphobles, olive flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, were exposed to oncomiracidia of H. okamotoi labelled with a fluorescent dye, 5- (and -6) carboxyfluorescein diacetate succinimidyl ester, and the numbers of parasites on the gills and skin were recorded at intervals. Oncomiracidia were attached to gills and skin of all the experimental fish species immediately after exposure, and the infection intensity on T. rubripes was higher than that on T. niphobles and much higher than those on the other two species. After 2 days, the attached parasites remained on the gills of T. rubripes, but disappeared from the other hosts. During in vitro experiments, gill filaments excised from seven different fish species (four fish species used in the in vivo experiments and panther puffer, Takifugu pardalis, southern flounder, Paralichthys lethostigma and spotted halibut, Verasper variegates) were exposed to oncomiracidia and the attachment to each fish species and subsequent larval behaviour was observed. The percentage of post-larvae that attached to T. rubripes was slightly higher than those which attached to congeneric fish species and much higher than those of non-tetraodontid fish species. In vivo and in vitro experiments demonstrated that oncomiracidia of H. okamotoi have an affinity for their natural host, T. rubripes, and congeneric fish species. The disappearance of attached post-larvae from 'alien' hosts within 2 days during in vivo experiments suggested that host recognition by oncomiracidia and subsequent post-larval survivability are involved in the host specificity of H. okamotoi.