A polyclonal CD3+4-8-WT31- cell line (termed SFG) was utilized for mice immunization in order to produce monoclonal antibodies (mAb) specific for the T cell receptor (TcR) gamma/delta. Hybrid supernatants were screened for their ability to induce SFG cells (but not conventional TcR alpha/beta + CTL lines) to kill the murine Fc receptor-positive P815 target cell line. Three hybrids, termed G1, A13 and F11, were isolated according to this screening. By indirect immunofluorescence G1 mAb reacted with 65% of SFG cells, while A13 stained 26% and F11 75% of cells. Double-fluorescence analysis revealed that G1 and A13 mAb identify two distinct, non-overlapping subsets of cells present in the SFG cell line. The reactivity of the mAb was also analyzed on a panel of representative TcR gamma/delta clones. G1 mAb reacted with 5 clones, that were also stained by the previously described BB3 mAb (recognizing the disulfide-linked form of TcR gamma/delta). These clones failed to react with A13 and delta-TCS-1 mAb (the latter of which is known to react with a non-disulfide-linked form of TcR gamma/delta). Out of six clones that reacted with A13 mAb, four were also delta-TCS-1+, whereas two were delta-TCS-1- and none of them reacted with G1, (or BB3) mAb. In contrast to the mAb above, F11 brightly stained the G1+A13- clones and more weakly the G1-A13+ clones. Moreover, F11 efficiently triggered both types of clones to kill the P815 target cells while G1 and A13 were able to trigger only G1+ or A13+ clones, respectively. None of the mAb above reacted with a large number of CD3+WT31+ clones. Antibody-induced surface antigen modulation experiments indicated that molecules recognized by G1, A13 and F11 were physically associated on cell surface with CD3 determinants. In addition, immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (performed on 125I-surface-labeled TcR gamma/delta+ clones) revealed that molecules recognized by G1, A13 and F11 displayed an apparent mol. wt. corresponding to that of CD3-associated TcR molecules, immunoprecipitated by anti-CD3 mAb from the same clones.