A variant of the Jurkat leukemia cell line termed JA3 (surface phenotype: T11+., T3+, T4-, T8-, T6-, T1+, 3A1+, HLA-DR-, Tac-, 4F2+) has been used for mouse immunization and production of monoclonal antibodies (MAbs) to molecules carrying clonotypic determinants that are thought to serve as receptor for antigen on human T cells. JA3 had been selected because of its ability to release large amounts of IL-2 following stimulation with phytohemagglutinin (PHA) or anti-T3 antibody in the presence of phorbolmyristate acetate (PMA). This functional property has been exploited for screening of MAbs potentially directed to idiotype-like structures of JA3 cells. Thus supernatants of hybridomas (obtained by fusing mouse splenocytes with P3-UI myeloma cells) were analyzed for their ability to induce JA3 cells to release IL-2 in the presence of PMA. Four stimulatory antibodies reacted with JA3 but not with polyclonal T-cell populations, T-cell clones, or T and B tumor cell lines as assessed by indirect immunofluorescence and fluorescence-activated cell sorter (FACS) analysis. All 4 antibodies immunoprecipitated the same disulphide-linked heterodimeric molecules having a molecular mass (Mr) of approximately 85,000 under non-reducing conditions that was resolved in 2 major peptides of 40,000 and 45,000 under reducing conditions. These data indicate that these antibodies (termed anti-JTi) were directed to the clonotypic-restricted structures of the JA3 T-cell-receptor molecules. Unlike the anticlonotypic antibodies described so far, anti-JTi MAbs were capable of triggering IL-2 production even in unbound, soluble form, in the absence of adherent cells or PMA. Competitive inhibition experiments, in which 35S-labelled anti-JTi MAbs have been used, provided evidence that they may be directed against different epitopes on the same clonotypic structure.