As the lymphotropism of hepatitis C virus (HCV) has already been ascertained, and in the light of the fact that the immune defense system is an organized network composed of functionally interrelated tissues, this study was carried out to verify the possible involvement of spleen in HCV-related chronic hepatitis. In this cross-sectional study we measured spleen longitudinal diameter by ultrasound, beta2-microglobulin serum levels and splenic artery resistivity index (SARI) by Doppler in 51 patients treated with standard combined (Peg-Interferon plus Ribavirin) antiviral therapy. Thirty-three patients (17 females) completed the regimen and were compared to 31 controls (16 females). The mean basal values of spleen longitudinal diameter were higher in patients with chronic hepatitis than in controls, i.e., 116 mm (9.4) versus 102.7 mm (9.3), P = 0.0001. In the same patients a significant trend towards increased spleen longitudinal diameter was found after antiviral therapy, independently of the stage of HCV-related chronic hepatitis. The median values of the beta2-microglobulin concentrations were not significantly higher in the patients with HCV-related chronic hepatitis compared to controls, i.e., 1.3 (0.5-2.6) versus 1 (0.6-1.4), P = 0.16, although during the course of therapy they were significantly increased. SARI values of HCV-related chronic hepatitis patients were different from those of controls, but were unvaried compared to values at the end of treatment. Neither spleen measurements nor serum beta2-microglobulin levels were able to predict therapeutic response to antiviral therapy. A stimulation/expansion of lymphoid tissue was found in patients with HCV-related chronic hepatitis. Such evidence raises the question whether physicians should continue to prescribe antiviral therapy in non-responders and supports the use of a new scheme (SLD plus beta2-MG) to diagnose this ongoing, apparently reversible, but nevertheless dangerous immunologic process.