The physical association of dendritic cells and T lymphocytes in clusters is required for primary immune responses. We have used oxidative mitogenesis as a model to examine the requirements for accessory cell-T-cell clustering and T-cell activation; in this polyclonal response, periodate-treated T cells cluster with dendritic cells (DC) and proliferate. Here we observed firstly that macrophages (M phi) but not B cells or B blasts could also cluster with periodate-treated T cells, but they did not stimulate proliferation. Thus, clustering with a cell that can express MHC class II (Ia) molecules and synthesize interleukin-1 (IL-1) is not sufficient for mitogenesis, and Ia expression is not sufficient for clustering. Secondly, that proliferation in oxidative mitogenesis and the allogeneic mixed leucocyte reaction (MLR) was inhibited by CD18, CD4 and CD8 antibodies but not, with one exception, by others of the panel tested. Thirdly, using rapid cluster assays we showed that DC and M phi formed temperature-dependent clusters with syngeneic and allogeneic periodate-treated T cells. Clustering was inhibited by CD18 antibodies, probably at the level of the T cell but not the accessory cell, and this may be a general feature of such inhibition. However, CD4 and/or CD8 antibodies did not affect clustering, showing that these molecules are involved in subsequent stages of T-cell activation. Since clustering of DC and M phi with periodate-treated T cells requires CD18, but only the former leads to proliferation, we conclude that CD18-dependent clustering is not sufficient for mitogenesis.