Objective:To detect HIV-1 infection in window period and estimate HIV-1 incidence among men who have sex with men(MSM). Methods:Pooled HIV-1 RNA RT-PCR(3-staged pooling by 20∶1,5∶1,1∶1) was conducted and HIV-1 RNA copies were quantitated using COBAS AMPLICOR HIV-11.5 version MONITOR Test. Results:The plasma samples from 948 MSM,which were negative by HIV third-generation antibody assay were tested. Four samples were found to be HIV-1 RNA positive,and acute infection was diagnozed with seroconversion during follow up. The HIV-1 incidence was 5.55% per year[95%CI,3.50%~7.59%]. Conclusion:Pooled HIV-1 RNA RT-PCR is a good method of testing,and can be applied to screen HIV infection in winow-period in high-risk groups.