We assessed hepatitis C virus (HCV) infection in 99 asymptomatic blood donors positive using a first-generation HCV antibody assay. When tested with second-generation assays, 86 (87%) donors were reactive (group 1), 2 (2%) were indeterminate (group 2), and 11 (11%) were non-reactive (group 3). Viraemia was revealed by polymerase chain reaction in all group 1 cases. The 2 group 2 cases and 6 (55%) group 3 cases were also viraemic. Viraemia was confirmed by a branched DNA assay in the 2 group 2 cases and 4 (36%) group 3 cases. Serum HCV RNA levels were further studied using a competitive reverse transcription-polymerase chain reaction assay. All cases in groups 2 and 3 were low viraemic (range 10(4)-10(5.5) copies/ml) compared with the 9 group 1 cases examined (range 10(7)-10(9) copies/ml). No correlation was evident between viraemic levels and antibody cut-off index in the first-generation assay. These findings indicate the possibility that low levels of viraemia can occur in individuals non-reactive in second-generation HCV antibody assays.