In Japan, between the end of December 2003 and March 2004, four outbreaks of acute, highly transmissible and lethal disease occurred in birds in three prefectures separated by 150-450 km, involving three chicken farms and a group of chickens raised as pets. The cause of each outbreak was an H5N1 influenza A virus-the first highly pathogenic virus to be isolated from the outbreaks in Japan since 1925. The H5N1 virus was also isolated from dead crows, apparently infected by contact with virus-contaminated material. These H5N1 viruses were antigenically similar to each other, but could be differentiated from other H5 viruses, including those isolated from Hong Kong in 1997 and 2003, by use of a panel of monoclonal antibodies in hemagglutination inhibition assays. Genetically, the H5N1 viruses in Japan were closely related to each other in all genes and were genetically closely related to a single isolate of genotype V that was isolated in 2003 in the Guandong Province of mainland China (A/chicken/Shantou/4231/2003). The virulence of the index isolate (A/chicken/Yamaguchi/7/2004) was studied in chickens and mice. Chickens intravenously or intranasally inoculated with the isolate died within 1 or 3 days of inoculation, respectively. In mice, although this virus replicated well in the lung without prior adaptation and spread to the brain, the dose lethal to 50% of the mice was 5 x 10(5) 50% egg infectious doses (EID50), which is less pathogenic than the Hong Kong 1997 H5N1 viruses isolated from humans. Our findings indicate that the H5N1 viruses associated with the influenza outbreaks in chickens in Japan were genotypically closely related to an H5N1 virus isolated from chicken in China in 2003 (genotype V), but were different from those prevalent in southeastern Asia in 2003-2004 (i.e., genotype Z) and that these highly pathogenic viruses can be transmitted to crows, which are highly susceptible to these viruses.