In addition to their functional role as peptide-binding proteins HLA class II Ag can also act as signal-transducing molecules. The present study showed that cross-linking of HLA class II Ag by the anti-HLA-DR mAb L243 or by the anti-HLA-DR,-DP mAb IVA12 significantly (p < 0.05) increased the release of TNF-alpha by the EBV-B lymphoblastoid cell line JY. In contrast, the anti-HLA-DR mAb 2.06 or the superantigens staphylococcal exotoxin toxic shock syndrome toxin-1 and staphylococcal enterotoxin B that bind to HLA-DR,-DQ Ag did not affect the release of TNF-alpha by JY cells. The accumulation of TNF-alpha in the culture medium of JY cells peaked at 24 h, decreased thereafter, and was found to be dependent on the dose of mAb L243 or mAb IVA12 used to cross-link HLA class II Ag. mAb L243 or staphylococcal exotoxin toxic shock syndrome toxin-1 enhanced the spontaneous homotypic aggregation of JY cells and mediated a dose-dependent inhibition of JY cell proliferation. These phenomena were not mediated by TNF-alpha released in response to cross-linking of HLA class II Ag; polyclonal anti-TNF-alpha neutralizing antibody did not affect JY cell aggregation and the inhibition of JY cell proliferation mediated by mAb L243. In contrast, TNF-alpha secreted by JY cells enhanced a nuclear factor-kB-like activity through the binding to the 75-kDa TNF-alpha receptor. These results demonstrate an additional role of HLA class II Ag as signal-transducing molecules regulating the production of bioactive TNF-alpha by EBV-B cells. The release of TNF-alpha after the triggering of HLA class II molecules could be relevant to different aspects of B cell biology and might play a role in the pathogenesis of human diseases in which antibodies cross-reactive to HLA class II Ag have been identified.