The ligand for CD40 (CD40L) is a protein which is expressed on CD4 T cells following their activation: CD40-CD40L interactions are absolutely required for the induction of T cell-dependent antibody responses, yet little is known about the mechanisms whereby CD40L+ primary T cells activate naive B cells, since the protein is only transiently expressed and is rapidly down-regulated following T cell-B cell contact. We show here, using a variety of assays, that co-stimulation of primary murine T cells via CD3 and CD28 stabilizes the expression of the CD40L protein. Firstly, T cells stimulated in this manner express higher levels of CD40L when activated in the presence of B cells, compared to CD3-activated T cells. Secondly, the CD40L expressed on CD28-co-stimulated T cells is more resistant to B cell-induced down-regulation. Finally, CD3/CD28-preactivated, rested T cells re-express higher levels of CD40L more rapidly following re-stimulation via CD3 than T cells preactivated via CD3 alone. CD3/CD28-preactivated T cells, but not CD3-activated cells, are competent to induce DNA synthesis in naive B cells, and this requires re-stimulation via CD3 and prolonged ligation of CD40. These data therefore reinforce the concept that naive T cells need to be activated initially by cognate interaction with B7-bearing antigen-presenting cells (such as dendritic cells), before becoming competent helper effector cells capable of driving B cells into proliferation via a CD40-dependent pathway.