T lymphocytes are divided into two subsets which express different T cell receptor heterodimers. In the peripheral blood of healthy individuals, the majority of T cells express the alpha/beta T cell receptor (> 90%) while a minority have the gamma/delta T cell receptor (< 10%). The gamma/delta T cells of adults use preferentially the V gamma 9V delta 2 chain combination. Although the stimulation requirements for gamma/delta T lymphocytes are still undetermined, it has been reported that gamma/delta T cells are not only stimulated, like alpha/beta T cells, by conventional protein antigens and superantigens, but also by unusual ligands. Mycobacteria selectively stimulate V gamma 9V delta 2 T cells, and a nonproteinacious low molecular weight fraction of 1-3 kDa has been identified as the tentative active component. Here, we confirm the nonproteinacious nature of this ligand, and show that it is comprised of unusual carbohydrate and phosphate. Importantly, cleavage of the terminal phosphate by alkaline phosphatase completely abrogates the stimulatory activity of the low molecular weight ligand for V gamma 9V delta 2 T cells. Even mycobacterial whole lysate loses its stimulatory activity, for this T cell subset, after dephosphorylation with alkaline phosphatase. These findings identify phosphocarbohydrates as a novel molecular entity with selective stimulatory activity for a defined T cell subset.