Serially collected serum samples from 81 patients with acute non-A, non-B hepatitis were tested for the presence of antibodies to hepatitis C virus (anti-HCV) by a second-generation enzyme immunoassay (EIA) test. Anti-HCV was detected in 56 cases (69%) during the first month, in 61 cases (75%) at 3 months and in 63 cases (78%) at 6 months. In those 18 patients showing anti-HCV negative results in the three determinations, hepatitis C virus (HCV) RNA was tested using a nested polymerase chain reaction (PCR) in the first serum sample and was detected in only one case. Anti-HCV or HCV-RNA positive episodes were considered as acute hepatitis C, while those negative for both markers were classified as acute non-A, non-B, non-C hepatitis. On comparing acute hepatitis C with the non-A, non-B, non-C episodes, no significant differences were found in the presence of jaundice, mean maximum alanine-aminotransferase (ALT) levels and positivity of markers of past hepatitis B virus (HBV) infection. However, patients with hepatitis C were significantly younger than those with non-A, non-B, non-C hepatitis (p = 0.002). Male sex (78.1% vs. 35.3%; p = 0.001), history of parenteral exposure (90.6% vs. 11.8%; p = 0.0001), and progression to chronicity (73.4% vs. 5.9%; p = 0.0001) were significantly more frequent in the HCV-related group. Although other possibilities cannot be excluded, these results suggest that there might be a different infectious agent implicated in the etiology of acute non-A, non-B, non-C hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)