Co-stimulation mediated by the CD28 molecule is considered critical in the activation of CD4+ T cells. In patients with rheumatoid arthritis and infrequently in normal individuals, CD4+ T cells lacking CD28 expression are expanded and contain clonogenic populations. To analyze whether these cells are independent of co-stimulatory requirements or whether they use co-stimulatory signals distinct from the CD28 pathway, we have compared CD4+ CD28+ and CD4+ CD28- T cell clones isolated from rheumatoid arthritis patients. Accessory cells supported the induction of CD25 expression as well as of proliferative responses after anti-CD3 cross-linking and prevented the induction of anergy in CD4+ CD28- T cell clones. In contrast to CD4+CD28+ T cells, the presence of accessory cells did not enhance the secretion of interleukin (IL)-2, interferon-gamma, or IL-4. The co-stimulatory signals did not involve CD28/CTLA-4-CD80/CD86 receptor-ligand interactions. The proliferative response of CD4+CD28- T cells could not be blocked by anti-CD2, anti-CD18, and anti-CD58 antibodies, suggesting that these receptor-ligand interactions cannot provide CD28- independent co-stimulation. Our data suggest that CD4+CD28- T cells require co-stimulatory signals for optimal induction of cell growth and CD25 expression as well as for the prevention of anergy. The co-stimulatory receptor-ligand interaction is independent of the CD28 pathway and may be involved in the oligoclonal expansion of the CD4+ CD28- T cell subset in rheumatoid arthritis.