The acquirement of resistance to rituximab has been observed in lymphoma patients. To further define the molecular basis for rituximab-resistance we have developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 structure at the protein and gene level, membrane reorganization, and signaling events following rituximab exposure. RRCL were generated by chronic exposure of Raji cells to escalating doses of rituximab alone (2R) or concurrently with human complement (4RH). Functional assays were performed to demonstrate decrease in rituximab-associated CMC and ADCC. Changes in the structure of CD20 were determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. Sequencing of the CD20 gene from Raji parental and RRCL was performed to determine differences. Following rituximab treatment, lipid raft domains were extracted from Raji and RRCL to determine changes in polarization. Redistribution of CD20 antigen were evaluated by Western blotting. No significant changes were observed in the expression of the external domain of CD20 antigen between rituximab-sensitive and RRCL as demonstrated by flow cytometric analysis and Western blotting. However, significant changes in the internal domain of CD20 were observed in RRCL. Specifically, changes in the N-terminal, and to a lesser degree the C-terminal region of the internal domain of CD20 were observed in RRCL. The CD20 gene sequence was found to be identical between Raji cells and RRCL, suggesting a post-transcriptional regulatory mechanism is responsible for the changes in the structure of CD20. Redistribution of CD20 into the lipid raft domains was more evident in Raji parental cells when compared to RRCL, and a decrease in p38 activation was observed following exposure of RRCL to rituximab. In conclusion, our data strongly suggests that the acquirement of rituximab-resistance is associated with significant changes in the structure of the internal domain of the CD20 antigen. The abnormal production of truncated forms of the CD20 antigen, while not affecting external rituximab binding resulted in changes in the redistribution of CD20 antigen into lipid raft domains and a decrease in signaling events. Post-transcriptional events leading to the acquirement of truncated forms of CD20 may impair signaling activation, thus contributing to rituximab-resistance seen in RRCL.