Activation of T cells requires co-stimulation, in addition to signals through the antigen-receptor complex. Antigen encounter without adequate co-stimulation results in T-cell desensitization or anergy, a mechanism of peripheral tolerance and an apparent obstacle to cancer immunotherapy. One important co-stimulatory pathway involves CD28 engagement by CD80 or CD86. However, other ligand-receptor pairs can also provide co-stimulation and may have important functions modulating the immune response. Previous reports indicated that co-stimulation using 4-1BB ligand (4-1BBL) or agonistic anti-4-1BB antibodies could prolong T-cell responses, avoid activation-induced cell death and promote anti-tumour responses in mice. To further investigate the potential for cancer immunotherapy, we studied the effects of CD80/CD86 and 4-1BBL in repeated stimulation of human T cells and asked whether 4-1BBL might be capable of reversing anergy. We expressed CD80, CD86 and 4-1BBL in A549 lung carcinoma cells using adenovirus vectors and co-cultured these with human T cells stimulated with anti-CD3 antibody. Proliferation co-stimulated by CD80 or CD86 was transient; however, 4-1BBL-co-stimulated cultures continued to proliferate for up to 5 weeks, with repeated stimulation. Combined co-stimulation with CD80/CD86 and 4-1BBL also allowed continuous proliferation at a faster rate than either signal alone. Co-stimulation with 4-1BBL did not suppress expression of the inducible, inhibitory CD80/CD86R, CTLA-4. Significantly, we show that T cells that had become non-responsive to anti-CD3, either alone or together with CD80/CD86 co-stimulation, and thus were anergic, could be reactivated to proliferate when costimulated with 4-1BBL, either alone or combined with CD80/CD86.