Thirteen T cell hybridomas and two cell lines expressing γδ TCR were generated from B10 and B6 CD3+CD4-CD8- splenic T cells. At least three different types of γ-chains (Vγ2-Cγ1, Cγ2, and Cγ4) were shown to be expressed in these cells. Analysis of Vδ gene segment usage in these hybridomas was performed by Northern blot hybridizations using probes specific for the Vγ1 through Vγ6 gene families and electrophoretic analysis of polymerase chain reaction amplified DNA obtained using V region-specific oligonucleotide primers for the Vδ gene families as well as for Vα10, Vα4, and Vα11. These analyses revealed that Vδ5 gene segments are used by a high percentage of splenic γδ CD4-CD8- T cells (53% of the cells in our panel). Other Vδ segments expressed by our panel of cells were Vδ2, Vδ4, Vδ6, and Vα10, indicating the Vδ repertoire expressed in the spleen is similar, but possibly not identical to the adult thymus repertoire. Sequence analysis of the V-D-J joinings of the δ-chain messages revealed substantial diversity, indicating that the δ-chain repertoire expressed in peripheral lymphoid organs uses a significant portion of the potential diversity predicted for these chains. These results demonstrate that, in spite of the low numbers of γδ T cells in peripheral lymphoid organs, the diversity of their TCR is extensive enough to play an important role in the immune response.