Information about the character and grade of the intrahepatic immune response in viral hepatitis is important for the evaluation of disease stage and effect of therapy. Complications like haemorrhage limit the frequent performance of tissue-needle biopsies (TB), and the cells of peripheral blood have to be used as surrogate markers instead. Fine-needle-aspiration biopsy (FNAB) of the liver represents a safe and atraumatic method that allows frequent cytological sampling. Our aim was to investigate whether flow cytometry of FNAB specimens allows co-analysis of phenotype, function and specificity of key populations of liver-infiltrating lymphocytes (LIL). In 20 consecutive patients with chronic viral hepatitis [10 hepatitis B virus (HBV), 10 hepatitis C virus (HCV)], flow cytometry was performed on FNAB cytology, and simultaneously on lymphocytes isolated from a TB and peripheral blood mononuclear cells (PBMC). The ratio of CD8+/CD4+ lymphocytes in FNAB correlated well with LIL from TB (r =0.78, P < 0.05) but differed from PBMC (mean ratio: 2.6, 2.1 and 0.7, respectively). Similarly, a correlation was observed for percentage CD56+ natural killer (NK) cells (mean %: 29.9, 32.3 and 14.5, respectively; r = 0.69, P < 0.05). The percentage of interferon (IFN)-gamma-producing CD3+ lymphocytes in both FNAB and TB was higher than in PBMC (mean %: 41, 44 and 22, respectively; P < 0.05). Furthermore, tetrameric complexes allowed analysis of HBV-specific T cells in FNAB specimens. In conclusion, flow cytometry of FNAB allows easy, atraumatic and reliable analysis of lymphocytes obtained from the intrahepatic compartment. Therefore, the FNAB is a valuable tool in the study of the immunopathology of viral hepatitis, and it may contribute to the improved clinical evaluation of chronic viral liver disease.