BACKGROUND: Little is known about the prevalence of serum hepatitis C virus (HCV) RNA in blood donors with HCV antibodies and persistently normal alanine aminotransferase (ALT) levels. STUDY DESIGN AND METHODS: Thirty-nine anti-HCV-positive donors with normal ALT on four determinations at 3-month intervals were further tested monthly for 6 months, and they had normal ALT values. The presence of HCV RNA was determined in these 39 donors. RESULTS: Serum HCV RNA was detected in 16 of 39 donors, 14 of 14 who reacted on second-generation recombinant immunoblot assay (RIBA-2) and 2 of 15 who were indeterminate. None of the 10 RIBA-2-nonreactive donors had evidence of viremia. The 15 RIBA-2- indeterminate samples were tested with third-generation RIBA (RIBA-3); the results showed reactivity in 5 (including the 2 HCV RNA positive), an indeterminate pattern in 7, and nonreactivity in 3 (all RNA negative). Among HCV RNA-positive subjects, mean age (p < 0.05), mean ALT (p < 0.001), signal-to-cutoff (S:CO) ratio on second-generation enzyme-linked immunosorbent assay (p < 0.001), and gamma globulin levels (p < 0.05) were higher than those among HCV RNA-negative subjects. During 6 additional months of ALT monitoring, completed by 36 of 39 donors, increased values were detected in 6 (5 HCV RNA positive). In 4 of those 6, however, ALT levels were less than 1.5-fold the upper normal limit. HCV RNA results were unchanged at the end of 1-year follow-up. CONCLUSION: Forty-one percent of anti-HCV-positive donors with persistently normal ALT had active HCV infection. Long-term ALT monitoring allowed the detection of significantly increased enzyme values in only 2 of 16 viremic donors. Reactivity on RIBA-2 or -3, greater age, mean ALT levels in the upper range of normal, higher S:CO ratio on second-generation enzyme-linked immunosorbent assay, and higher gamma globulin levels were predictive of viremia.