Immunotherapy with rituximab (chimeric anti-CD20 monoclonal antibody, Rituxan), alone or in conjunction with chemotherapy, has significantly improved the treatment outcome of lymphoma patients. Via an elusive mechanism, a subpopulation of patients becomes unresponsive and/or relapses. To recapitulate various aspects of acquired resistance, rituximab-resistant (RR) clones were established from lymphoma lines and compared with parental cells. Surface CD20 expression was diminished in the clones. The clones neither responded to rituximab-mediated growth reduction or complement-dependent cytotoxicity nor underwent apoptosis in response to cross-linked rituximab. Rituximab failed to chemosensitize the RR clones, which exhibited constitutive hyperactivation of the nuclear factor-kappaB and extracellular signal-regulated kinase 1/2 pathways, leading to overexpression of B-cell lymphoma protein 2 (Bcl-2), Bcl-2-related gene (long alternatively spliced variant of Bcl-x gene), and myeloid cell differentiation 1 and higher drug resistance. Unlike parental cells, rituximab neither inhibited the activity of these pathways nor diminished the expression of resistant factors. Pharmacologic inhibitors of the survival pathways or Bcl-2 family members reduced the activity of these pathways, diminished antiapoptotic protein expression, and chemosensitized the RR clones. These novel in vitro results denote that continuous long-term rituximab exposure culminates in RR clones that do not respond to rituximab-mediated effects, have altered cellular signaling dynamics, and exhibit different genetic and phenotypic properties compared with parental cells. The data also reveal that although RR clones exhibit higher resistance to rituximab and cytotoxic drugs, these clones can be chemosensitized following treatment with pharmacologic inhibitors (e.g., dehydroxymethylepoxyquinomicin, bortezomib, PD098059) that target survival/antiapoptotic pathways. The findings also identify intracellular targets for potential molecular therapeutic intervention to increase treatment efficacy. The significance and potential clinical relevance of the findings are discussed.