The polymerase chain reaction (PCR) technique is a powerful new tool for amplifying target DNA, thus allowing for sensitive detection of specific nucleic acid sequences. One important potential use of PCR involves screening the donated blood supply for transfusion-transmitted viruses. Realization of this goal has been limited by (1) the requirement for multiple, discrete PCR reactions to amplify and detect target sequences of more than one virus, and (2) the lack of a rapid, nonhazardous means for specific detection of one or more PCR-amplified products. We report the simultaneous amplification of three distinct target sequences without discernable loss in sensitivity toward any single target sequence. We also demonstrate very rapid separation and detection of PCR-amplified viral DNA through the use of automated capillary DNA chromatography. Amplified DNA peaks were initially identified by scanning the capillary effluent at ultraviolet wavelengths, while discrimination of human immunodeficiency virus type 1 and human T-cell leukemic virus type I PCR-amplified DNA was accomplished through use of virus-specific, fluorescently labeled primers and probes. These results indicate progress toward an automated system for screening the blood supply for nucleic acid sequences of multiple pathogens.