To determine the etiologic agents of two atypical pneumonia human cases in Hunan Province in 2005-2006 and to study their pathogenic potential, the patients' respiratory tract samples and sera were collected. The respiratory tract samples were tested by real-time RT-PCR and RT-PCR methods, and the sera by hemagglutination-inhibition assay. Virus was isolated from case 2 and its genome was sequenced and analyzed. Results showed the H5 nucleic acid tests of two cases were positive. The H5-specific antibody titer of the convalescence serum of case 1 showed a 4-fold greater rise than that of the acute phase. And case 2's antibody titer of acute phase was negative. The two atypical pneumonia cases were confirmed as the avian influenza A (H5N1) infection cases. Viral strain A/Hunan/1/2006 was isolated from case 2. Phylogenetic and molecular analysis suggested that 8 gene segments of A/Hunan/1/2006 originated from avian viruses. And A/Hunan/1/2006 was similar with viruses isolated from avian in Hunan Province. The isolated virus did not recombine with human influenza viruses and no obvious variation was observed.