The pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) has been linked with the production and activity of certain growth factors. Tumor necrosis factor (TNF-alpha) is important for the growth and survival of B-CLL cells. TNF-alpha promotes the proliferation of the malignant cell clone and is believed to play a role in the progression of B-CLL. The aim of our study was to examine the level of production and intracellular expression of TNF-alpha by T-and B-cells in B-CLL in correlation with stage of disease and clinical parameters. Using a three-color flow cytometry technique we analyzed intracellular TNF-alpha expression by B-cells (CD19+) and by T-cell subsets (CD3+/CD4+ and CD3+/CD8+) in peripheral blood (PB) and bone marrow (BM) from 40 patients with B-CLL and 24 healthy controls. A higher number of TNF-a-positive B-cells were found in PB and BM in B-CLL patients than in normal controls. In BM this difference was statistically significant (p< 0.007). Likewise, in PB and BM the percentage of T-cells expressing TNF-alpha was significantly higher in B-CLL patients than in normal controls (PB: p< 0.00001; BM: p< 0.007). CD3+/CD4+ cells from patients displayed a lower level of intracellular TNF-alpha expression than did CD3+/CD8+ cells (PB: p< 0.0001; BM: p< 0.04). The number of T-cells expressing TNF-alpha in B-CLL patients was higher in those with stages III-IV than in patients with early stage disease (PB: p< 0.007; BM: p< 0.01). Additionally, PB and BM T-cell subsets from patients in stages III-IV had a statistically significant higher level of cytoplasmic TNF-a expression than the corresponding cells from healthy controls (PB: p< 0.02; BM: p< 0.05). In PB the percentage of CD4+ and CD8+ T-cells expressing cytoplasmic TNF-alpha positively correlated with the stage of disease, total tumor mass (TTM) score and lymphocytosis. In BM only the percentage of CD8 T-cells positively correlated with TTM score and lymphocytosis. The expression of TNF-alpha in leukemic B-cells did not correlate with any progression parameters of disease. The results obtained suggest that malignant B-cells are exposed to large numbers of T-cells able to synthesize and secrete TNF-alpha. Moreover, T-cells even though fewer than B-cells may be an important source of TNF-a in advanced stages of disease. This indicates that the TNF-alpha can be associated with progression of B-CLL and may be implicated in some side-effects associated with this disease.