A second generation recombinant immunoblot assay to detect antibodies against Hepatitis C virus (RIBA II, Ortho Diagnostic Systems) was applied to 30 serum samples repeatedly reactive to ELISA anti-HCV c100-3, 11 from hemodialysis patients, 11 from patients with chronic hepatitis and 8 from patients with cirrhosis. The assay detects individual antibodies directed to 4 antigenic components of the C virus, 2 antigens, c100 and 5-1-1 contained in the original ELISA assay and 2 new antigens, c33c and c22-3 the latter structural in nature. From the total of 30 samples, 23 (77%) were reactive, 4 (13%) indeterminate and 3 (10%) non reactive to the RIBA II assay. Much variation was observed regarding the response to each individual antigen. c22-3 was the most frequently reactive with the highest intensity. RIBA II assay confirmed a high percentage of the reactive results with the ELISA c-100-3, serum samples that were non reactive to RIBA showed a low optical density with ELISA. c22-3 was the most sensitive antigenic component.