CD4(+) T-cell responses are important for the outcome of hepatitis C virus (HCV) infection. However, the functional status of HCV-specific CD4(+) T cells in persistent infection is poorly understood. It is generally recognized that proliferative capacity of HCV-specific CD4(+) T cells is weak or absent in persistent infection, but whether this results from deletion of antigen-specific cells or represents maintenance of antigen-specific but poorly proliferative populations is not defined. We used a set of ex vivo assays to evaluate the functionality of HCV specific CD4(+) T cells in persistent and resolved infection. Peripheral blood mononuclear cells (PBMC) from 24 prospectively recruited HCV polymerase chain reaction (PCR) positive individuals, 12 spontaneously resolved individuals (i.e. anti-HCV+, PCR-) and 11 healthy controls were analysed for interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) secretion by enzyme linked immunospot assays (ELISpot). HCV-specific CD4(+) proliferative responses of carboxy fluorescein succinimidyl ester-labelled PBMC were assessed using a sensitive single cell flow cytometric assay. Sustained IFN-gamma ELISpot responses were observed in the PCR+ group. However, proliferation of HCV-specific CD4(+) T cells in the PCR+ group was substantially reduced on a per cell basis, in parallel to IL-2 secretion, compared with responses in the PCR- group. In PCR- individuals, a strong relationship between cytokine secretion and proliferative capacity was seen. However, in PCR+ individuals, IFN-gamma secretion far exceeded proliferative capacity. During persistent HCV infection, some CD4(+) T-cell specificities appear to be lost, as measured using a range of techniques, but others, potentially important, are maintained as IFN-gamma secretors but with low proliferative capacity even using a highly sensitive assay. Such subsets may yet play a significant role in vivo and also provide a template for modulation in immunotherapeutic interventions.