Previous studies of the human TCR-delta gene identified a single commonly used V delta segment, denoted V delta 1. To better understand the extent of the human TCR-delta V gene repertoire, TCR-delta transcripts and gene rearrangements were examined in a new panel of cloned human TCR-gamma/delta lymphocytes. Through this analysis we identified and determined the structures of two new V delta segments, denoted V delta 2 and V delta 3. These V delta segments are different from previously characterized V alpha segments, supporting the notion that the human V delta and V alpha repertoires are distinct. Examination of V gamma gene segment usage in these cells reveals that the V delta 2 gene segment is used in conjunction with the V gamma 2 gene segment. Blot hybridization indicates that the V delta 2 gene segment lies between V delta 1 and D delta-J delta-C delta, and within 100 kb of the latter. Analysis of genomic clones indicates that the V delta 3 gene segment lies in an inverted orientation, approximately 2 kb 3' of C delta. This implies that rearrangement of V delta 3 to D delta-J delta-C delta occurs by inversion. Together with previous mapping studies, these results indicate that human V delta segments are dispersed, rather than clustered, within the TCR-alpha/delta locus. The analysis of rearrangements in polyclonal thymocyte DNA suggests that there may be a limited number of additional V delta gene segments yet to be characterized.