Cytotoxic T cells (Tc) can be primed in vitro to specifically kill autologous Epstein-Barr virus (EBV) transformed lymphoblastoid cell line cells (LCL). The antigen recognized by Tc on the LCL cells is designated LYDMA (lymphocyte determined membrane antigen). Recognition of LYDMA may also require simultaneous recognition of HLA determinants. Despite its immunogenic potency, the genetic origin and molecular structure of LYDMA remains undetermined. Most LCL cells do not produce EBV virus, but all LCL cells appear to express an immunogenic, non-HLA class I, 47,000 Dalton determinant which is detected by several independently derived mouse monoclonal antibodies. This determinant is not readily detected on peripheral blood lymphocytes, nor on EBV-associated Burkitt's lymphoma cell lines. We examined the potential relationship between this 47,000 MW Epstein-Barr virus associated cell surface antigen (the EBVCS antigen) and LYDMA. Populations of Tc generated by in vitro activation with irradiated autologous LCL cells were potent at destroying EBVCS bearing autologous LCL cells. The addition of anti-EBVCS antibody to this cytotoxic interaction had no suppressive effect. Furthermore, single cell derived, LYDMA reactive, Tc clones able to specifically destroy autologous LCL were studied. Cytotoxicity could be inhibited by monoclonal antibody to HLA-Class I determinants, but not by monoclonal antibodies to EBVCS. Thus, these LYDMA reactive Tc clones do not recognize the same antigenic determinant as the monoclonal antibodies specific for EBVCS. It remains uncertain whether this 47,000 MW glycoprotein that carries the serologically detected EBVCS antigen may be directly recognized by human T cells.