In recent years, a highly pathogenic avian influenza virus (AIV) H5N1 subtype has emerged as a pathogenic agens for a variety of species including humans. To investigate the pathogenicity of AIV, the isolate ZFE/2004 (H5N1) was used to infect chickens, mice, and pigs. The results showed that the isolate showed attributes of the highly pathogenic AIV such as high pathogenicity index (intravenous pathogenicity index (IVPI) of 2.85 and intracerebral pathogenicity index (ICPI) of 3.00) and a specified hemagglutinin (HA) cleavage site. In vivo experiments indicated that this isolate was highly lethal to the Balb/c mice. In addition, it was able to infect pigs, elicit specific antibodies, and cause "flu-like" clinical symptoms. Typical pathological changes were observed in the chickens as well. In vitro experiments showed that the isolate ZFE/2004 was able to induce apoptosis of infected MDCK cells that was detected by the DNA fragmentation analysis and electron microscopy.