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.