We have previously reported that r28M, a recombinant bispecific single-chain antibody directed to a melanoma-associated proteoglycan (NG2) and the costimulatory CD28 molecule on T cells, induced T-cell activation, which resulted in tumor-cell killing. T-cell activation did not require a primary signal through the T-cell antigen receptor (TCR)/CD3 complex and depended on the presence of NG2-positive tumor cells. Here, we further investigate this phenomenon of a target cell-restricted, supra-agonistic CD28 stimulation with bispecific antibodies. To this end, we exchanged the NG2 targeting part of r28M with a single-chain antibody directed to the B-cell associated antigen CD20. The resulting bispecific single-chain antibody, termed r2820, induced supra-agonistic T-cell activation, which required the presence of autologous normal or malignant B cells, respectively. Once activated, T cells were capable of destroying lymphoma target cells.These findings demonstrate that supra-agonistic CD28 stimulation with bispecific single-chain antibodies is a robust and readily reproducible phenomenon. In the context of experimental tumor therapy, it may provide a valuable alternative to the unrestricted T-cell activation induced by 'super-agonistic', monospecific CD28 antibodies.