Individual nucleic acid-amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce the transfusion transmission of human immunodeficiency virus (HIV) and hepatitis C viruses. This screening test can identify donations made during the immunological window period before seroconversion. The impact of this technology in our blood donors and transfusion routine was studied. In all, 47 866 donations were tested from March 2004 until November 2005, according to Brazilian legislation, using two approved enzyme immunoassays for HIV antibodies and individual NAT. Supplemental tests included Western blot, p24 antigen detection and quantitative PCR-HIV-1. Among the donors screened, two (one first-time and one repeat donor) were non-reactive in enzyme immunoassays, with negative confirmatory p24 antigen and Western blot, but positive for HIV-1 NAT. Although serological analysis for HIV is a primary tool for diagnostic testing, the addition of NAT allowed for identification and prevention of component transfusion from two HIV-positive blood donations during an 18-month period. The screening of donors reduced the immunological window period, permitting the identification of very early stage HIV infections. In addition, this report also emphasized the fact that the risk of HIV transmission is not limited to the first-time donors.