Although the spring season in Japan is cold, some fly species are present. However, no studies have been reported on the possible role of flies in transmission of H5N1 influenza virus. Therefore, an entomological survey was conducted in March 2004 to investigate the possibility of blow flies transmitting H5N1 virus, using flies collected from around the infected poultry farm in Tamba Town for virus detection and isolation. Blow fly collection was carried out on 10-11 March 2004, just after the H5N1 outbreak at poultry farm B [11] . A sunny place protected from strong wind was selected, and rotten fish bait was placed on the ground. A total of 926 flies were collected within a 2.3 km radius of poultry farm A in Tamba Town ( Figure 2 ), representing eight fly species with >80% of the collected flies identified as either Calliphora nigribarbis Vollenhoven or Aldrichina grahami (Aldrich) (Figure 3 ). Influenza A virus matrix protein (M) and hemagglutinin (HA) genes were detected in the intestinal organs, crop, and gut of C. nigribarbis and A. grahami by reverse transcriptionpolymerase chain reaction (RT-PCR) [11] . The prevalence of H5 subtype virus (20-30%) was higher in flies of both species collected 600-700 m from poultry farm A and lower (10%) in flies collected >2 km from poultry farm A. We found that nearly 5% of C. nigribarbis collected around the affected areas contained infectious H5N1 viruses. viral M, HA, and NA genes were amplified by PCR with universal primers, full-length sequences were analyzed, and sizes were found to be 991, 1,707, and 1,362 bp, respectively. These sequences showed high similarity to those of strains from chickens (A/chicken/Kyoto/3/2004) and crows (A/crows/Kyoto/53/2004) isolated during the 2004 outbreaks in Kyoto, with >99.9% identity for all three genes. The virus from C. nigribarbis (A/blow fly/Kyoto/93/2004) was characterized as an H5N1 subtype influenza A virus based on neuraminidase gene (NA) sequences. In addition, the HA1-HA2 connecting peptide sequence in the HA gene segment was RERRRKKR↓G. Finally, virus isolated from C. nigribarbis was characterized as a highly pathogenic H5N1 subtype influenza A virus.