To elucidate the developmental pattern and diversity of murine cluster of differentiation (CD)3-associated TCR-γδ heterodimers, adult and fetal thymocytes were examined for cell-surface expression of various γ- and δ-encoded TCR. Biochemical analysis, using antisera specific for distinct Cγ gene products, revealed the presence of T cells expressing Cγ1 and/or Cγ4 heterodimers in adult and fetal CD4- CD8- thymocyte populations. Although CD4- CD8- thymocyte populations express both Cγ1 and Cγ4 TCR-γδ heterodimers early in fetal thymus development, the relative level of Cγ4-expressing T cells was significantly lower than previously observed in peripheral lymphoid organs. In addition, biochemical studies revealed the presence of TCR-γδ heterodimer(s) expressed during fetal ontogeny which were not detected in adult thymocyte or peripheral lymphoid populations. Studies of N-glycosylation patterns of one of these heterodimers suggested that it contained a rearranged Vγ3/Cγ1 gene product. To examine in detail individual TCR-γδ heterodimers, a panel of TCR-γδ expressing hybridomas was prepared. Biochemical analysis at the clonal level revealed that indeed three distinct TCR-γδ heterodimers were present at day 16 of fetal thymus development, with TCR-γ-chains most likely encoded by Vγ2/Cγ1, Vγ3/Cγ1, and Vγ/Cγ4. Together these findings suggest an ordered development of TCR-γδ T cells in the thymus and selective expression of distinct TCR-γδ subsets in peripheral lymphoid organs such as spleen and lymph nodes.