The clinical significance of single band reactivity (indeterminate pattern) at anti-hepatitis C virus (HCV) second-generation recombinant immunoblot assay (RIBA-2) was investigated in symptomless subjects with normal liver function tests to obtain data for their counseling and clinical management. Serum and hepatic HCV RNA were determined by the nested polymerase chain reaction, and liver histology was evaluated in 40 symptomless blood donors with stable indeterminate RIBA-2 pattern, including 38 reactive to c22-3. All but one had normal alanine aminotransferase (ALT) levels. Two new immunoblot tests, RIBA-3 and INNO-LIA HCV Ab III (LIA-III), which incorporate additional HCV antigens, were also done to assess whether they could identify the viremic subjects. Ten cases (25%, confidence interval 12 to 38) were HCV RNA positive. Three of the HCV RNA-positive and none of the HCV RNA-negative subjects had chronic hepatitis. RIBA-2 strong intensity of reaction (score >2+) was observed in all the HCV RNA-positive and in 12 HCV RNA-negative subjects. RIBA-3 and LIA-III gave positive results in 9 of 10 and 10 of 10 HCV RNA-positive, but also in 8 of 30 and 24 of 30 HCV RNA-negative subjects. A c-22-3 reactivity score of 4+ by RIBA-3 and E2/NS1 reactivity by LIA-III were both strongly associated with HCV RNA (P < .001). Based on relatively high prevalence of chronic hepatitis in our series (30%), apparently healthy subjects with stable indeterminate RIBA-2 pattern and HCV RNA positivity should be considered for liver biopsy independently of ALT profile. Reactivity to particular HCV antigens by third-generation tests helped to identify the subjects with current infection. (HEPATOLOGY 1995; 21:913–917.)