An increased frequency of CD5+ B cells (or, according to a new nomenclature, B1 cells) has been detected in the peripheral blood of a proportion of patients with myasthenia gravis (MG), as in some other autoimmune diseases. To elucidate the pathogenic significance of this B-cell subset in myasthenia gravis, mononuclear cells from the peripheral blood of six MG patients were separated into T and B lymphocytes by a magnetic cell separation procedure employing superparamagnetic microbeads (MACS). Subsequently, the B-cell fraction was depleted of CD5+ B cells in a second separation. The resulting purified CD5- B-cell fraction was cultured alone or with the addition of autologous T cells. Anti-acetylcholine receptor (AChR) synthesis by CD5- B cells in cultures with T cells was significantly increased by pokeweed mitogen (176 +/- 130 fmol/ml per week/2 x 10(5) B cells) compared with unfractionated cells (75 +/- 101) or CD5- B cells alone (19 +/- 4). These results demonstrate that in MG anti-AChR are synthesized, at least in part, by CD5- B cells which are dependent on T cells. Although this does not exclude the existence of AChR-specific CD5+ B cells, it provides evidence against a pivotal role of this B-cell subset in anti-AChR synthesis.