Clinical trials of monoclonal antibodies in combination with chemotherapy have reported previously unattained response rates in CLL because this approach reduces disease burden to levels detectable only by flow cytometry or molecular methods. The latest studies from CALGB have demonstrated that rituximab added concurrently or sequentially to fludarabine (Flu) for symptomatic, untreated CLL allows to achieve higher remission rates, longer progression free and overall survival. Nevertheless, the analysis of different biologic parameters could better explain the discordant outcome independent of treatment observed in these studies. Recent literature data indicate that unmutated VH genes, CD38 and/or ZAP-70 protein tyrosine kinase overexpression may predict both a lower response and a shorter survival. We performed at our Institution a phase II study that added rituximab sequentially to Flu as initial therapy for symptomatic, untreated CLL in order to evaluate either the toxicity or the clinical response or outcome. Complete remission (CR) was also assessed by a multiparametric flow cytometric method. ZAP-70 protein and CD38 antigen were determined before chemotherapy on mononuclear cells by flow cytometry using an anti-ZAP-70 and an anti-CD38 antibody, respectively. Forty-nine B-CLL patients, median age 59 years (range 37–74) received six monthly courses of Flu (25 mg/m2 for 5 days) and four weekly doses of rituximab (375 mg/m2) starting on an average of thirty days (range 5–180) after completion of the Flu therapy. According to modified Rai stages, 4 pts had a low stage, 42 an intermediate stage and 3 a high stage. Three out of 49 pts experienced fever, chills and rigors, during the first infusion of rituximab and only 1 patient presented grade 3 infective lung toxicity according to WHO. Hematologic toxicity included neutropenia (grade 1 and/or 2 in 12 pts, grade 3 and/or 4 in 22 pts), thrombocytopenia (grade 1 and/or 2 in 4, grade 3 and/or 4 in 3 pts) and anemia (grade 2 in 3 pts). Based on the NCI criteria, 45/49 (91.8%) pts achieved a CR, 3/49 (6.1%) a partial remission (PR) and 1/49 (2%) no response (NR). The median follow-up duration was 29 months. Median duration of CR and PR has not been reached. Noteworthy, our B-CLL pts treated sequentially with Flu and rituximab experienced a very long progression-free survival (PFS) from treatment (72% at 3 years). ZAP-70 and CD38 were positive (>20%) in 19/48 (39.5%) and in 13/48 (27%) pts, respectively. Minimal residual disease (MRD) performed on bone marrow by flow cytometry was positive (>5% CD19+CD5+CD79b- CLL cells) in 7/37 (19%) analysed pts. A significant shorter PFS was observed in ZAP-70+ pts (38% vs 100% at 3 years; P=0.003), in CD38+ pts (39% vs 92% at 3 years; P=0.007) and also in pts with higher MRD after treatment (50% vs 81% at 2 years; P=0.04). Therefore, the addition of monoclonal antibodies, such as rituximab, to chemotherapy allowed a better outcome in CLL, exerting a key role to eradicate MRD. Moreover, the stratification of pts within different risk classes using novel biologic predictive factors, such as ZAP-70 and CD38, might allow us to offer more tailored treatment strategies, reserving experimental approaches and/or transplantation procedures only to CLL subsets with proved adverse biologic and clinical features.