A B cell line, B6-1710, expressing murine AIDS virus superantigen stimulates T cell hybridomas derived from a Ld-specific CTL clone to produce IL-2 regardless of their CD4/CD8 phenotype. However, B6-1710 cell did not stimulate the original CTL clone, L3, in either proliferation or cytolytic assays. Both B6-1710 and Ld+ P815 cells stimulated a significant Ca2+ influx in the T cell hybridomas. In contrast, only P815 stimulated tyrosine phosphorylation of a 19-kDa protein in the T cell hybridoma. The addition of the nonspecific protein kinase C activator PMA restored the proliferative response of L3 cells to B6-1710. PMA did not induce the lysis of B6-1710 cells by L3. These results suggest that murine AIDS virus encoded superantigen elicits a TCR-mediated signal different from that caused by nominal Ag. This different signaling by viral superantigen may be important for the development of viral pathogenesis in vivo.