Lymphoid cells in most patients with chronic lymphocytic leukemia (CLL), when treated with rituximab, become CD20-. This is thought to be due to masking of CD20 by rituximab. We used specific antimouse immunoglobulin antibodies to detect rituximab on the surface of CLL lymphocytes and we demonstrate that rituximab is rarely detectable after therapy. Only 3 of 65 patients with CLL had rituximab detectable on their lymphocytes after rituximab therapy despite the fact that most had no detectable CD20 expression. In vitro mixing of CLL or Raji cells with rituximab demonstrated that rituximab was detectable on the surface of cells due to its binding to CD20. However, the addition of plasma led to the down-modulation of CD20 expression, and the rituximab became undetectable. This down-modulation of CD20 protein expression was associated with a down-modulation of CD20 mRNA. CLL cells that lost their CD20 expression regained CD20 expression after 24 hours in culture. These data suggest that rituximab therapy leads to a substantial but transient down-modulation of CD20 expression and that negativity for CD20 in cells from patients treated with rituximab is not necessarily due to CD20 masking. The importance of this down-modulation in the efficacy of current therapy with rituximab needs further investigation.