Pemphigus is a life-threatening autoimmune bullous disease associated with autoantibodies to desmoglein 1 and/or 3. The anti-CD20 chimeric mouse monoclonal antibody rituximab has previously been successfully applied in more than 130 reported pemphigus patients with severe and/or refractory disease. Since antibodies against other therapeutics such as IFNalpha and beta, erythropoietin, and TNFalpha antagonists had led to decreased efficacy of these drugs, we determined anti-rituximab antibodies in 11 patients with pemphigus before rituximab administration as well as 3, 9, and 15 months thereafter. For this purpose, a novel, affinity capture elution assay was established using rabbit IgG against the F(ab)2 fragment of rituximab. In addition, serum levels of rituximab were determined by a competition ELISA. In 2 of 11 pemphigus patients, antibodies to rituximab were detected. In both patients, only a partial remission was observed under treatment. In addition, when followed over a longer period of time, the occurrence of anti-rituximab antibodies paralleled an increase in disease activity. Of the 9 patients without development of antiantibodies to rituximab, in 5, all lesions healed and in 4, partial remissions were seen. These observations show that antibodies to rituximab are generated in some patients during rituximab treatment and may be associated with a less favourable response to treatment.