Abstract The chimeric monoclonal antibody (mAb) rituximab has previously been shown to induce cell death upon binding surface CD20 of normal and malignant B-cells by three distinct and complementary mechanisms: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and direct apoptosis. The contribution of each of these mechanisms to the clinical efficacy of rituximab observed in lymphoma patients has yet to be fully elucidated. While laboratory and clinical data supports a role for CDC, ADCC, and/or direct signaling in rituximab activity, a better understanding of individual mechanisms and their interaction is necessary to develop strategies to improve rituximab’s biological activity or overcome resistance. Several previous studies have demonstrated that rituximab has the ability to induce lipid raft domain (LRD) polarization and stabilization of CD20 in LRDs. The contribution of LRD re-organization to complement activation following rituximab binding to surface CD20 was studied in rituximab-resistant B-cell NHL cells and MOLT-4 T-cell lymphoma cells transfected with wild-type and truncated forms of CD20. Rituximab-resistant cell lines (RRCL) were generated from well characterized B-cell lymphoma cell lines (Raji, RL, SU-DHL-4) by exposing them to increasing concentrations of rituximab or rituximab plus human serum as a source of complement. Individual clones were then isolated by limiting dilution from RRCL and the parental cell lines from which they were derived. Standard 51Cr-release assays were used to measure CDC-associated cell lysis following exposure of cells to rituximab in the presence of human serum. Following repeated exposure to increasing concentrations of rituximab (alone or in the presence of human serum) most clones were found to be significantly more resistant to rituximab-induced CDC than parental cells from which they were derived. Rituximab-sensitive cells were found to rapidly polarize CD20-containing lipid raft domains following rituximab binding whereas resistant cells demonstrated significant impairment of this process. Upon chemical disruption of lipid rafts in sensitive cells, the ability of rituximab to induce CDC was significantly reduced. Additionally, MOLT-4 T-cells transfected with truncated forms of CD20 were shown to have a reduced ability to polarize into lipid rafts following anti-CD20 binding and to have a significant loss of rituximab-induced CDC. Taken together these data suggest that lipid raft polarization following rituximab binding to wild-type CD20 is a required event in the induction of CDC and that impairment of lipid raft polarization is a potential mechanism contributing to rituximab resistance.