Human γδ T cells recognize prenyl pyrophosphate Ags and their analogues in a Vγ2Vδ2 TCR-dependent manner. Few data are available regarding the TCR structural requirements for recognition of such prenyl pyrophosphate Ags by γδ T cells. Presently, we made chain pair switch, chimeric, and site mutant γδ TCRs and transfected them into TCR− mutant Jurkat T cells to examine the effects of changing the TCRγ junctional region sequences on reactivity to prenyl pyrophosphate Ags. Substitution of the TCRγ junctional region (N and J) sequences from an Ag-reactive TCR with TCRγ junctional region sequences from an Ag-nonreactive TCR abrogated reactivity to the prenyl pyrophosphate Ag isopentenyl pyrophosphate and to its synthetic analogue ethyl pyrophosphate but not to a mycobacterial supernatant containing a mixture of prenyl pyrophosphate Ags. Substitution of only the TCRγ N nucleotide region with that from this Ag-nonreactive TCR destroyed reactivity to isopentenyl pyrophosphate and to the mycobacterial supernatant. Substitution of the entire Vδ2 chain from the Ag-reactive TCR with a Vδ1 chain from an Ag-nonreactive TCR yielded a prenyl pyrophosphate Ag-nonreactive TCR. Thus, using TCR mutagenesis and TCR transfectants, we show that γδ TCR reactivity to prenyl pyrophosphate Ags is dependent upon the junctional region of the TCRγ chain and upon pairing of Vγ2 and Vδ2 TCR chains. These structural requirements of TCRγδ recognition of prenyl pyrophosphates distinguish this reactivity from that of protein superantigens and emphasize the importance of the TCRγ CDR3 loop and adjacent residues.