Rhesus monkeys with high specific H5N1 antibody were inoculated the second time with H5N1 virus, the result of the second time H5N1 inoculation and the effect of first time H5N1 inoculation on second inoculation was evaluated. Monkeys of NO. 3, NO. 4, NO. 5 were inoculated with H5N1 allantoic fluid and NO. 6 with noninfectious allantoic fluid by intratracheal thyrocricoid puncture. Three months later, NO. 4, NO. 5, NO. 6 monkeys were infected with 7 ml TCID50 10(4.875) H5N1 allantoic fluid and NO. 3 monkey with 7 ml noninfectious allantoic fluid at the same time by the same method. Clinical symptoms were recorded and antibody response was detected by ELISA. NO. 3, NO. 4, NO. 6 monkeys were killed after 72 h post infection and NO. 5 monkey was killed after 7 days post infection. Pathologic changes of the infected monkeys' lung were examined by HE staining,immunohistochemistry and the virus in lung was detected by RT-PCR. Results showed that NO. 3, NO. 4, NO. 5 monkeys still retained high level of specific antibody, H5N1 virus only could be detected in NO. 6 monkey's lung by immunohistochemistry and RT-PCR ,and the lung of NO. 6 monkey injured worst . It can be concluded that Rhesus monkeys inoculated with H5N1 avian influenza A virus at the first time could retain a high level of specific antibody in 90 days and the clinical symptom had almost recovered, the ability of Rhesus monkeys to resist second infection of H5N1 virus was enhanced notably at that moment.