(E)-1-Hydroxy-2-methyl-but-2-enyl 4-diphosphate, a recently discovered intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis, has been shown to act as a potent immunomodulator. In cultures of human peripheral blood mononuclear cells from eight non-related donors, the compound stimulated the proliferation of Vgamma9/Vdelta2 T lymphocytes with a median EC(50) of 70 pM when 10 U/ml of IL-2 was used as costimulant. Isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP) and some structural analogs of (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate also stimulated Vgamma9/Vdelta2 T-cell proliferation, albeit at much higher concentrations. The Vgamma9/Vdelta2 T-cell proliferation is highly dependent on the seeding density used in culture. All phosphoantigens tested elicited the proliferation of two T-lymphocyte populations with different apparent ratios between the expression level of Vdelta2 and Vgamma9 chains.