Human V gamma 9V delta 2 T cells were shown recently to respond to nonpeptidic phosphorylated molecules of mycobacterial origin (previously referred to as TUBag). To investigate the early events of V gamma 9V delta 2 T cell activation, we have analyzed induction of cytotoxicity and TNF production of T cell clones by these molecules. We showed that within minutes after exposure, TUBag induced cytotoxicity of V gamma 9V delta 2 CTL (but not of CTL expressing other TCR V gamma/V delta or V alpha/V beta regions) against a broad set of target cells, including effector cells themselves. Induction of V gamma 9V delta 2 cytotoxicity by TUBag was blocked by anti-TCR mAbs and was abrogated after dephosphorylation of TUBag. Similarly, TUBag, but not dephosphorylated TUBag, induced massive TNF production by V gamma 9V delta 2 T cell clones only, which already was significant 20 min after exposure. Of note, only basal amounts of TNF were produced when cells were maintained in suspension in the presence of TUBag, indicating that efficient activation of TNF production induced by these compounds required a cell-to-cell contact. Finally, preincubation experiments allowed us to demonstrate that activation of V gamma 9V delta 2 T cells was strictly dependent on the presence of TUBag because preincubation of the targets with TUBag followed by a single wash abrogated the activation. Taken together, these results strongly suggest that activation of V gamma 9V delta 2 cells by TUBag occurs after binding of these compounds to (a) yet unidentified, highly conserved, and broadly distributed molecule(s). The results also suggest either that TUBag induces a very rapid and transient expression of a V gamma 9V delta 2 TCR ligand or, more likely, that TUBag is a low affinity component of a complex recognized by the V gamma 9V delta 2 TCR.