The introduction of the anti-CD20 monoclonal antibody, rituximab, into the treatment of patients with B-cell lymphomas has improved the overall response rate, as well as the response duration and the overall survival of these patients. However, only a few studies have addressed the question of whether higher CD20 expression parallels with better treatment outcomes. The aim of this study was to assess the relationship between the level of CD20 expression and overall survival (OS), disease-free survival (DFS) along with the overall response rate (ORR) in B-cell lymphoma patients. The ultimate objective of the study was to determine the cut-off value of CD20 expression together with the predictive significance of better outcome of rituximab treatment. One hundred and fourteen patients with different histological types of B-cell lymphomas treated with rituximab and chemotherapy between 2003 and 2007 were enrolled in the study. All patients had CD20 expression assessed prior to the beginning of treatment. The level of CD20 expression was determined by quantitative flow cytometric measurements, while the OS and DFS were evaluated by means of Kaplan-Meier survival curves. The cut-off value of CD20 expression, which predicts a better response to rituximab in patients with B-cell lymphomas, was determined at 25.000 molecules of equivalent soluble fluorochrome (MESF). Our data show that patients who achieved complete response after rituximab therapy had a significantly higher expression of the CD20 antigen (p=0.018) than those whose disease only stabilized after rituximab therapy. No significant difference was observed in the response duration between the patients with CD20 antigen expressed above the cut-off value and those expressing CD20 antigen below the cut-off value [hazard ratio (HR), 0.5667; 95%CI, 0.124 to 3.18, p=0.57]. Even though we have proved that patients with a CD20 expression level above the cut-off value treated with rituximab had a significantly longer OS [hazard ratio (HR), 0.4573; 95%CI, 0.1364 to 0.9461, p=0.0383] than patients with a CD20 expression level below the cut-off value. Among our study population, 17.5% had a CD20 expression level below the cut-off value. The highest percentage (80%) of the patients with a CD20 expression level below the cut-off value belonged to the group of chronic lymphocytic leukemia (CLL) patients, while the lowest (6.7%) was observed in the follicular lymphoma (FL) patient group. These data indicate that a higher level of CD20 expression correlates with an improved OS in patients treated with rituximab. The cut-off limit of CD20 expression suggested to have the predictive significance of better outcome was in our series set at 25.000 MESF. This cut-off value should be considered when the decision regarding treatment with rituximab is taken. However, these results warrant further studies on larger groups of patients.