The antigen presenting cell (APC) activity of uninduced, resident peritoneal macrophages and B cells was compared to that of antigen-specific B cell hybridomas by measuring proliferative responses of antigen-specific, MHC-restricted T cell clones. The results demonstrate that peritoneal macrophages and B cells are much more efficient APC than irradiated splenic filler cells, and that unirradiated B cells were as good as, if not better than, macrophages. Both B cells and macrophages can be pulsed with antigen, although pulsed B cells were always found to be more efficient than pulsed macrophages. However, the APC activity of B cells was exquisitely sensitive to irradiation. The relative contribution of macrophages and B cells to the APC activity of mixed populations was easily distinguished by complement dependent lysis with monoclonal antibodies specific for unique differentiation antigens expressed by these cells. Normal peritoneal macrophages and B cells present the synthetic terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) to GAT-specific T cells clones and beef insulin to insulin-specific T cell lines nonspecifically. The APC activity of antigen-specific B cells was also examined by using novel GAT-specific, nonsecretor B cell hybridomas produced by fusing GAT-primed spleen cells to the HAT sensitive Balb/c lymphoma, M12.4.5. The hybridomas selected for these studies were GAT-specific, sIg+, Ia+ cells. These hybridomas presented GAT to GAT-specific T cells more efficiently than heterogeneous B cells suggesting that interaction with surface Ig receptors facilitated the uptake and/or processing of antigen. GAT-specific B cell hybridomas, like normal B cells, presented soluble beef insulin to an insulin-specific T cell clone nonspecifically. However, after pulsing with antigen overnight, the GAT-specific B cell hybridoma could activate only GAT-specific T cells.