The mouse T-cell receptor (TCR) alpha/delta locus was mapped using 17 V alpha and 4 V beta subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The alpha/delta locus spans about 1 Mb. The distance between the 3'-most V gene segment (V delta 1) and the delta constant gene (C delta) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual V alpha gene segment subfamilies are dispersed throughout the locus. In contrast, the V delta gene segments V delta 1 to 5 are clustered at the 3' end of the V gene segment cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the alpha/delta locus and on organization features that might influence the expression of specific V gene segments in gamma delta cells.