We have examined the ability of several class II-positive tumor cell transfectants to stimulate murine Th1 clones. Most of the transfectants failed to activate the Th1 clones and, in fact, induced Ag-specific anergy. However, we found that one tumor, a UV-induced fibrosarcoma (6130-VAR1), was capable of stimulating both cytokine production and proliferation in Th1 clones. We believe that 6130-VAR1 cells possess a unique costimulatory activity for the following reasons. First, these cells fail to express known costimulatory molecules including B7-1 and B7-2. Second, 6130-VAR1-mediated stimulation of Th1 clones was not blocked by anti-CD28 Fab or by CTLA4Ig, which suggests that members of the B7 family were not up-regulated during the course of stimulation and that activation does not occur via a CD28-dependent pathway. Third, 6130-VAR1 could provide costimulation when presented on a different surface than the class II/peptide ligand for the TCR. This last finding suggested that the activity on these cells was not simply an adhesion molecule that facilitated increased efficiency of T cell:MHC interactions. Finally, like B7-1 transfectants, stimulation by class II-positive 6130-VAR1 cells prevented the induction of anergy in the Th1 clones. Taken together, these results strongly suggest that 6130-VAR1 expresses a unique costimulatory activity (VAM-1) that, like B7-1, can promote T cell activation and prevent anergy induction.