T suppressor (Ts) hybridomas were produced by fusion of the III/4 T cell hybridoma with splenic T cells from CBA mice tolerized with subimmunogenic doses of bovine serum albumin (BSA). Both the Ts hybridoma cells and a suppressor factor (TsF) inhibited in an antigen-specific and I-Ak-restricted fashion the in vitro proliferative response of BSA-immunized lymph node cells. In addition to the suppressive activity, the hybridoma lines displayed spontaneous cytotoxicity against various tumor targets. The isolation of Ts subclones that are suppressive but not cytolytic, as well as the existence of the noncytolytic TsF, indicates that suppression of antigen-specific T cell proliferation is not dependent on cytolytic activity. The Ts hybridomas were I-A restricted, as are many T helper cells. Therefore, a potential similarity with respect to antigen receptor genes was expected. Southern blot analysis with a probe specific for genes encoding the beta chain of the T cell receptor on T helper and T killer cells revealed no rearrangement of the beta genes in the Ts cells. The data imply that neither the antigen receptor on the I-A-restricted Ts cells nor the receptor involved in the cytolytic interaction with tumor targets use the same beta chain constant region as T helper and T killer cells.