It is becoming apparent that γδ T cells form an important part of the adaptive immune response. However, the ligands recognized by γδ T cell receptors (TCRs) and the exact biological function of the cells that express this receptor remain unclear. Numerous studies have shown that the dominant human peripheral blood subset of γδ T cells, which express a Vγ9Vδ2 TCR, can activate in response to low molecular weight nonpeptidic molecules. Some of these components have been purified from bacteria or parasites. We examined the activation of polyclonal γδ T cell lines, clones with Vγ9Vδ2 and Vγ9Vδ1 TCRs, and γδ T cells directly ex vivo in response to multiple phosphate, alkylamine and aminobisphosphonate (nBP) antigens and purified protein derivative from Mycobacterium tuberculosis (PPD). Vγ9Vδ2 T cells were able to respond to multiple small organic molecules of highly variable structure whereas cells expressing a similar Vγ9 chain paired with a Vδ1 chain failed to recognize these antigens. Thus, the TCR δ chain appears to make an important contribution to the recognition of these antigens. The kinetics of responses to alkylphosphate and alkylamine antigens differ from those of responses to the nBP pamidronate. These different classes of antigen are believed to have differed mechanisms of action. Such differences explain why nBPs can be pulsed onto antigen presenting cells (APCs) and still retain their ability to activate γδ T cells while alkylphosphate and alkylamine antigens cannot. We also demonstrate that a substantial proportion of the cells that produce IFNγ directly ex vivo in response to PPD are γδ T cells and that γδ T cell activation requires contact with cells of human origin.