In the current study, we confirmed previous findings suggesting that gamma delta T cells were involved in the successful adoptive cell transfer of contact sensitivity (CS) by alpha beta CS-effector T cells. In this study, we used hamster anti-mouse gamma delta-TCR mAb treatment of CS-effector T cells, followed by enrichment and removal of the gamma delta T cells with goat anti-hamster Ig-linked magnetic beads, or by addition of hemolytic rabbit C. This removal of gamma delta T cells abrogated adoptive cell transfers of CS, despite the presence of alpha beta T cells that are known to mediate CS. FACS analysis documented enrichment of gamma delta T cells rising from 1 to 2% of the starting cells, to 60 to 95% of the magnetic bead adherent cells. Adoptive cell transfer of CS was reconstituted by adding back to the alpha beta cells, highly enriched gamma delta cells attached to anti-gamma delta-TCR magnetic beads. Not only were gamma delta-enriched T cells from sensitized mice able to assist immune CS-effector alpha beta T cells, but gamma delta T cells from normal nonimmune mice also had CS-assisting activity, and furthermore, neither were MHC-restricted in this function. Thus, CS-assisting gamma delta T cells were present endogenously in normal mice without prior immunization, and acted without Ag specificity and without MHC restriction, to assist CS-effector alpha beta T cells. Similar studies, with hamster mAbs specific for V gamma and V delta portions of gamma delta-TCR, demonstrated that the gamma delta T cells that assisted the CS-effector alpha beta T cells preferentially expressed V gamma 5 and V delta 4 in their TCR. PCR analysis on extracted mRNA showed that V gamma 5 and V delta 4 gene segments indeed were rearranged and expressed in the sensitized and normal lymph nodes; and one-and two-color FACS analysis of magnetic bead-fractionated cells suggested that V gamma 5 and V delta 4 were expressed on the same T cells. In summary, these results demonstrated that V gamma 5+, V delta 4+, gamma delta T cells were needed to assist alpha beta effector T cells in the adoptive cell transfer of CS.