The ability of different subpopulations of blood mononuclear cells to serve as accessory cells in the activation of CD4+ and CD8+ T cells via Ti-CD3 or with phytohaemagglutinin (PHA) was studied. Pure CD4+ or CD8+ T cells did not respond to particle-bound anti-CD3 monoclonal antibodies (MoAb) or PHA, whereas responses were seen when non-T cells served as accessory cells. Removal of class II-positive cells from peripheral blood mononuclear cells (PBMC) or from non-T cells diminished, but did not completely abolish, the responses in both T cell subsets, indicating that the accessory cells are mainly found among the class II-positive cells. However, the class II molecules themselves were not involved, as demonstrated in antibody-blocking experiments. Removal of monocytes decreased the ability of non-T cells to serve as accessory cells for both CD4+ and CD8+ cells in PHA activation. In contrast, the removal of monocytes resulted in an enhanced activation by anti-CD3 MoAb in CD4+ T cells, while the activation of CD8+ T cells was less affected. Positively selected B cells were effective accessory cells in anti-CD3 and PHA activation. Furthermore, Epstein-Barr virus (EBV)-transformed B cell lines were very potent accessory cells both in anti-CD3 and PHA activation of T cells, and showed the strongest accessory cell function observed in this system on a per cell basis.