Despite the identification of hepatitis C virus (HCV) and the detection of anti-HCV antibodies in the serum of infected individuals, a sizeable proportion of patients who develop transfusion-associated acute non-A, non-B hepatitis following surgery do not develop anti-HCV antibodies. The cause of this disease remains unknown. To assess the role of homologous blood transfusion in anti-HCV-positive and -negative, non-A, non-B hepatitis following surgery, patients receiving homologous blood, autologous blood alone, or no transfusions were prospectively studied. Consumption of potentially hepatotoxic drugs was also quantified. Anti-HCV antibodies were tested retrospectively when commercial assays became available. Of the 181 patients who received homologous blood which tested negative for surrogate markers of infectivity, 19 (10.5%) developed non-A, non-B hepatitis, associated with anti-HCV seroconversion in three cases. Of the 90 autologous blood recipients, non-A, non-B hepatitis developed in one (1.1%), who did not seroconvert to anti-HCV. Of the 64 untransfused patients, non-A, non-B hepatitis developed in one (1.6%), who was anti-HCV-positive before surgery. Logistic regression analysis showed that the occurrence of non-A, non-B hepatitis was associated with homologous blood transfusion, but not with the consumption of potentially hepatotoxic drugs. The 16 homologous-blood recipients who developed anti-HCV-negative, non-A, non-B hepatitis had received blood from 70 donors, none of whom had detectable anti-HCV antibodies but six of whom had minimal elevations of serum aminotransferase activity. Anti-HCV-negative, non-A, non-B hepatitis is mainly transfusion-transmitted in the surgical setting. Known hepatotropic agents may be involved despite the absence of usual serum markers, but our results are also consistent with the involvement of an unidentified non-A, non-B, non-C agent.