An antigen-specific T-T hybridoma was mutagenized with ethylmethane sulfonate and negatively selected by anti-Ly-6 antibody-induced growth inhibition. One of the mutants generated, M4/8, had lost surface expression of a T cell receptor (TcR) V beta 8 epitope detected on the surface of the parental cell line. However, the mutant cell line did express high levels of TcR heterodimer as detected with a pan-specific anti-TcR antibody. CD3 epsilon, Ly-6 and Thy-1 were expressed at levels similar to the wild-type parental cell line. Analysis of the surface TcR/CD3 complexes by immunoprecipitation and two-dimension gel electrophoresis confirmed that the major discernable difference between the wild-type and mutant TcR/CD3 complexes resided in the TcR beta chain. The parental cell line had the potential to express two TcR heterodimers, V alpha V beta 1 and V alpha V beta 8, as determined by Northern blot analysis. Co-modulation experiments suggested that both types of receptors were expressed. However, the V alpha V beta 8 receptor was the predominant form. In contrast, the mutant M4/8 cell line did not synthesize V beta 8 mRNA and, thus, only the V alpha V beta 1 TcR was synthesized. Despite the normal surface expression of TcR/CD3 complex, the M4/8 mutant cell line did not produce interleukin 2 (IL 2) in response to antigen or soluble anti-CD3 epsilon monoclonal antibody (mAb). Furthermore, it responded poorly to concanavalin A, phytohemagglutinin and anti-Ly-6 mAb. Cross-linking of the stimulatory antibodies partially restored the IL 2 response to anti-CD3 epsilon or anti-Ly-6 to wild-type levels. Phorbol ester and ionomycin stimulated a full IL 2 response in the M4/8 cell line, demonstrating that the defect in the decreased signaling in the mutant did not result from a defect in the IL 2 gene program. In conclusion, these data suggested that the pairing of alpha/beta heterodimer not only determined antigen/MHC specificity but also the signaling efficiency of the TcR/CD3 complex.