Nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) of blood donated voluntarily after serological screening was implemented on July 1st 1999 for transfusion and plasma fractionation by the Japanese Red Cross blood transfusion services. From February 1st 2000, HBV, HCV and HIV-1 NAT screening of pools of 50 negative serologically screened donated blood was started and the results were reported within 1 day after blood donation. Systems were established for rapid shipment, electronic communication, automated specimen preparation, pooling and automated amplification and detection. At present, NAT screening is carried out within 1 day after donation. This report describes the blood screening system by NAT and the results obtained from over 16 million blood samples using simultaneous screening for HBV, HCV and HIV-1 with multiplex reagent. Between February 1, 2000 and December 31, 2002, 16012175 serologically negative units were tested by NAT. 308 units with Hepatitis B virus DNA (HBV DNA) were detected. The sensitivity of 50 pool NAT screening with input volume of 0.2 ml is significantly higher than that of highly sensitive HBsAg testing. 46 cases with HCV RNA and six cases with HIV-1 RNA were detected. These cases were not detected by HCV antibody and HIV-1 antibody screening. The false positive rate was 0.18%. The NAT system was developed from serological screening test negative non-remunerated voluntary donations. We supply blood products to medical organizations after screening by NAT for HBV, HCV and HIV-1 for transfusion and source plasma for fractionation. This is the first automated integrated system for prevention of transfusion transmitted HBV, HCV and HIV-1 infections, by NAT screening.