V delta 3 usage and combinatorial expression of V gamma and V delta regions was studied on peripheral T cells with a novel V delta 3-specific mAb (p11.10b), generated against a soluble V gamma 9V delta 3 TCR. V delta 3+ cells represented the vast majority of V delta 1/V delta 2- gamma delta T cells within peripheral blood and mucosal lymphocytes. No preferential V gamma region expression was noted within V delta 3+ cells, but the frequency of V gamma 9+ cells was significantly lower among V delta 3+ than among V delta 1+ or V delta 2+ PBL. Phenotypic analysis of cultured V delta 3+ cells sorted with p11.10b mAb revealed the presence of T lymphocytes with unusual phenotypes. First, cells carrying two distinct surface TCR delta-chains, recognized by both V delta 1- and V delta 3-specific mAbs, were detected in most T cell lines, though at frequencies much lower than that of dual gamma expressors, indicating that allelic exclusion of delta genes is more tightly regulated than that of gamma genes. Moreover, a significant fraction of V delta 3+ cells were recognized by C beta- but not C delta-specific mAbs. Molecular analysis of V delta 3+C beta+ clones revealed the presence of V delta 3J alpha C alpha transcripts in all of them. Given the peculiar location of the V delta 3 gene between the delta Rec/psi J alpha elements, those observations formally demonstrate that activation of rearrangements with J alpha elements is not necessarily preceded by a delta Rec/psi J alpha-mediated deletion of the delta locus on the same chromosome.