A hypersensitive bioassay of nucleopolyhedrovirus infecting a silkworm, Bombyx mori, (BmNPV) has been developed. Peroral administration of a chitin synthesis inhibitor, Polyoxin AL wettable powder (Polyoxin AL), to a silkworm resulted in a decrease of the median lethal concentration (LC₅₀) of BmNPV. The LC₅₀ in the silkworm ingesting Polyoxin AL decreased as the silkworm developed from the 2nd to the 4th stadium. LC₅₀ in a 2nd stadium silkworm ingesting Polyoxin AL was lower than that in a silkworm refrigerated at 4°C. Silkworms at the 2nd stadium that had ingested Polyoxin AL were used to detect BmNPV in the dust from 15 sericultural farmhouses. Dust samples were collected at a leaf storage space, a rearing room and a mounting room just before the beginning of silkworm rearing after the formalinisation of the rearing environment. The dust was collected again at the same places immediately after harvesting the cocoons. In addition to the BmNPV detection in the dust, occurrence of innerside-stained cocoons was examined at the same 15 farmhouses where the dust was collected. The following features of BmNPV contamination are recognized. In some farmhouses, BmNPV was not inactivated even after treatment with formalin. BmNPV accumulated in only one cycle of the silkworm rearing of about 3 weeks in almost all the farmhouses. Of all three places in which the dust was assayed, the mounting room tended to be most heavily contaminated by BmNPV. More innerside-stained cocoons occurred in a farmhouse where more silkworms died of BmNPV in an assay of the dust collected before the beginning of the silkworm rearing. On the other hand, these features of BmNPV contamination were not detected using silkworm larvae that had not ingested Polyoxin AL. This new bioassay for BmNPV utilizing Polyoxin AL-treated larvae is an effective tool to assess the cleanliness of the sericultural environment.