T cell hybridomas were generated from CD3+ CD4-, CD8-, splenocytes and fetal thymocytes. Vγ1-expressing proteins present on these murine TCR-γδ hybridomas were identified by using an anti-TCR Vγ1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR Vγ-Cγ4 chains and 31-kDa TCR Vγ-Cγ1/2 chains from distinct heterodimers expressed on the TCR-γδ T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-γ chain hybridized with a Vγ1 probe but failed to hybridize with a Vγ2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-γ chain hybridized with a Vγ2 probe. This 32-kDa TCR-γ chain was not immunoprecipitated by the anti-Vγ1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a Vγ1 to Cγ2 rearrangement, whereas the 32-kDa protein was the product of a Vγ2 to Cγ1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a Vγ1.2-Jγ2 rearrangement, and all three of the 41-kDa TCR-γ chains were the results of the Vγ1.1-Jγ4 rearrangements. This was the first demonstration at the clonal level of TCR-γ proteins which use members of the Vγ1 gene family, as well as the Cγ2 constant region. Additional biochemical analyses of the TCR-γ and -δ proteins from three independently derived Cγ4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral Cγ4-containing heterodimers may be contributed by the TCR-δ chains.