Cytokine expression assessed by flow cytometry in 53 acquired aplastic anemia patients before and after combined immunosuppression (EBMT WPSAA protocols) showed that CD3(+) marrow cells containing TNF-alpha, IFN-gamma and IL4 were similar in subjects with disease at onset (DO) and responsive to treatment who had more CD3(+)/TNF-alpha(+) and CD 3(+)/IFN-gamma(+) cells than normal controls. In vitro block of TNF-alpha and/or IFN-gamma significantly increased BFU-e over baseline in 28 patients. In responsive to treatment patients only TNF-alpha block significantly incremented colonies over normal controls. Absolute marrow CD3(+)/TNF-alpha(+) and CD3(+)/IFN-gamma(+) cells prospectively tested in a group of 21 subjects declined significantly more in Responders than in Non Responders to immunosuppression at Response Evaluation Time respect to Diagnosis. Both in Responders and in Non Responders these cells remained higher than in normal controls. This study suggests that immunosuppression does not fully clear excess TNF-alpha and IFN-gamma from marrow of patients with good outcome and raises the hypothesis that additional cytokine blockade might be useful in immunosuppression for acquired aplastic anemia.