Expanded CD4(+)CD28(null) T cells have not been described in the circulation of patients with chronic hepatitis B (CHB). The aim of the present was to detect and characterize the surface phenotype and functional capacity of these cells in CHB patients. Expanded CD4(+)CD28(null) T cells were detected in the circulation of CHB patients with high viral load and elevated aminotransferase levels. Most CD4(+)CD28(null) T cells showed a CD27-CD45RA(-)CD45RO(+) surface phenotype. The markers CD56, CD57 and killer immunoglobulin-like receptor (KIR) were detected on CD4(+)CD28(null) T cells, but the majority were positive for CD57. Functionally, CD4(+)CD28(null) T cells were found to be potent cytotoxic T lymphocytes with perforin and granzyme B secretion profiles. These findings indicate that the expanded CD4(+)CD28(null) T cells are cytotoxic memory T cells and display a distinct functional phenotype in comparison with CD4(+)CD28(+) T cells. The presence of these cells appears to be associated with inflammatory conditions, suggesting that these elevated CD4(+)CD28(null) T cells might be involved in the pathogenesis of CHB.