The diagnosis of acute hepatitis C virus (HCV) infection relies on documented positive-seroconversion of HCV antibody (anti-HCV). Because of the detection of seroconversion at an earlier stage by second or third generation anti-HCV enzyme immunoassays (EIA), the diagnosis of acute hepatitis C (AHC) may be underestimated. The aim of this study was to evaluate whether rising anti-HCV titre could be used to diagnose AHC or not. Eighteen patients with a clinical diagnosis of acute hepatitis C were enrolled, including eight cases with documented seroconversion to anti-HCV and 10 cases with clinically suspected acute hepatitis C. Four chronic hepatitis C patients with acute exacerbation were selected as a control group. Serial sera were assayed with a third generation anti-HCV (AxSYM, version 3.0; Abbott, Chicago, IL, USA) and recombinant immunoblot assays (RIBA; Chiron HCV 3.0 Strip; Immunoblot, Emeryville, CA, USA) and the titre of anti-HCV expressed as signal/cutoff (S/CO) ratio and the RIBA patterns were correlated. Seven of eight documented AHC (one lacking the initial serum) and five of 10 clinically suspected AHC showed a rising pattern of S/CO values. The initial S/CO values on the first visit were less than 40 in 14 of 18 cases. The RIBA pattern shifted from negative/indeterminate to positive in five of seven documented AHC and 4 of 10 clinically suspected AHC cases. Fifteen of 18 cases had seroconversions of at least one antibody, whilst 85.7% showed a rising S/CO ratio. On the contrary, the S/CO ratio and RIBA pattern remained unaltered in chronic hepatitis C with acute exacerbation. The rise in S/CO was usually accompanied with an increase in the number of RIBA reactive bands and their intensity in acute hepatitis C patients. The rise in S/CO ratios using a third generation anti-HCV assay and the RIBA pattern might be used as a supplemental diagnostic criterion for acute HCV infection.