Many different pathogens stimulate cells bearing the Vγ9-Vδ2 T cell receptor (TCR), which represent the most abundant population of human γδ cells. The antigens responsible for the stimulation of these γδ cells are not well characterized. Here, we describe six non-peptidic molecules which share this property: isopentenylpyrophosphate, dimethylallylpyrophosphate, 2,3-diphosphoglyceric acid, glycerol-3-phosphoric acid, xylose-1-phosphate, and ribose-1-phosphate. All these molecules are naturally occurring metabolites in prokaryotic and eukaryotic cells, and stimulate freshly isolated γδ cells from peripheral blood of different donors as well as established γδ clones. Comparison of their structure with that of similar but inactive molecules showed that both the number and position of the phosphate groups, as well as the residues connected with the carbon backbone are required for stimulation. The CD3-TCR complex is involved in cell triggering as shown by inhibition with anti-CD3 Fab fragments. However, all γδ clones were broadly cross-reactive and we could not isolate cells specific for only one ligand. The capacity of this frequent subset of γδ cells to recognize common bacterial metabolites confers the advantage to react rapidly to different invading pathogens.