Two subsets of human gammadelta T cells can be identified by T cell receptor (TCR) V gene usage. Vdelta2Vgamma9 T cells dominate in peripheral blood and recognize microbe- or tumour-derived phosphoantigens. Vdelta1 T cells are abundant in mucosal tissue and recognize stress-induced MHC-related molecules. Toll-like receptors (TLRs) are known to co-stimulate interferon-gamma (IFN-gamma) production in peripheral blood gammadelta T cells and in Vdelta2Vgamma9 T cell lines. By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated gammadelta T cells by TCR versus TCR plus TLR3 stimulation. Furthermore, we have investigated TLR expression in freshly isolated Vdelta1 and Vdelta2 subsets and cytokine/chemokine production in response to TLR1/2/6, 3 and 5 ligands. TLR1,2,6,7 RNA was abundantly expressed in both subsets, whereas TLR3 RNA was present at low levels, and TLR5 and 8 RNA only marginally in both subsets. Despite abundant RNA expression, TLR1 was rarely detectable by flow cytometry. In contrast, TLR2 and TLR6 proteins were detected in purified Vdelta1 and Vdelta2 T cells, and TLR3 protein was detected intracellularly in both subsets. TLR1/2/6, 3 and 5 ligands co-stimulated the IFN-gamma and chemokine secretion in TCR-activated Vdelta1 and Vdelta2 subsets, although the levels of IFN-gamma secreted by Vdelta1 T cells were much lower than those produced by Vdelta2 T cells. Our results reveal comparable expression of TLRs and functional responses to TLR ligands in freshly isolated Vdelta1 and Vdelta2 T cells and underscore the intrinsically different capacity for IFN-gamma secretion of Vdelta1 versus Vdelta2 T cells.