Recently, blood centers began investigational testing for HIV RNA by pooled nucleic acid testing (NAT). A 35-year-old frequent platelet donor tested HIV p24 antigen positive, antibody negative before implementation of NAT. He made 2 platelet donations (day –4 and –11) immediately before testing positive for HIV. The donor’s HIV seroconversion was monitored, and stored samples were tested retrospectively for HIV RNA. Platelet recipients were tested for HIV infection. The day –4 sample tested positive for HIV RNA by pooled and individual sample NAT. The day –11 sample tested negative for HIV RNA by both NAT tests. The 2 recipients of the day –4 platelets tested HIV RNA and p24 antigen positive. The recipient of the day –11 platelets could not be tested because he had died. HIV NAT would have prevented transmission of HIV had it been available at the time of this donor’s HIV seroconversion.