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It has previously been reported that singular reactivity against NS5 in the third generation hepatitis C virus (HCV) confirmatory recombinant immunoblot assay (RIBA-3) is not associated with detectable HCV RNA in sera. In order to investigate the significance of indeterminate HCV RIBA-3 results with particular regard to NS5, 165 sera with indeterminate RIBA-3 results were analysed for the presence of HCV RNA. Sera from blood donors constituted 58 of the 165 samples, whereas 11 were from immunocompromized patients. The remaining 96 sera were from non-immunosuppressed clinical patients. Of the 107 RIBA-3 indeterminate samples from clinical patients, 19 were HCV RNA positive (18%), with 3 of the 57 (5%) NS5 reactive samples having detectable HCV RNA. None of the 58 indeterminate samples obtained from blood donors had detectable HCV RNA. Thus, having an indeterminate RIBA-3, including singular reactivity against NS5, may be associated with the presence of detectable levels of HCV RNA in clinical patients but not necessarily in blood donors.