We have used gene-targeted mutation to assess the role of the T cell receptor delta (TCR delta) enhancer (E delta) in alphabeta and gammadelta T cell development. Mice lacking E delta exhibited no defects in alphabeta T cell development but had a severe reduction in thymic and peripheral gammadelta T cells and decreased VDJ delta rearrangements. Simultaneous deletion of both E delta and the TCR alpha enhancer (E alpha) demonstrated that residual TCR delta rearrangements were not driven by E alpha, implicating additional elements in TCR delta locus accessibility. Surprisingly, while deletion of E delta severely impaired germline TCR delta expression in double-negative thymocytes, absence of E delta did not affect expression of mature delta transcripts in gammadelta T cells. We conclude that E delta has an important role in TCR delta locus regulation at early, but not late, stages of gammadelta T cell development.