We produced and characterized a panel of anti-HLA class II monoclonal antibodies (mAb) to study the inhibition of T cell-dependent B cell differentiation. The newly produced mAb belonged to different isotypes and subclasses, and recognized at least two different nonpolymorphic HLA class II determinants. We found that anti-HLA class II mAb inhibited the pokeweed mitogen (PWM)- and interleukin 2-driven T cell-dependent IgM synthesis, but did not affect the phorbol myristate acetate-, phytohemagglutinin-, anti-T3- or PWM-induced T cell proliferation. Moreover, when the mAb were tested in a monocyte-independent B cell differentiation assay in which T cells are activated by insoluble anti-T3 mAb in the absence of accessory cells, no effect was seen on IgM synthesis. These data indicate that inhibition of T cell-dependent B cell differentiation by anti-HLA class II mAb is caused by interference with an accessory cell function essential for B cell differentiation.