Murine cytolytic T lymphocyte (CTL) clones specific for type A influenza virus antigens were generated by in vitro stimulation with syngeneic virus-infected cells in the presence of T cell growth factor (TCGF). All CTL clones recognize viral determinants shared by PR8 and X31 influenza viruses in association with a class I antigen, coded either by the H-2K or H-2D end of the appropriate haplotype. All clones express the Lyt2 antigen marker. Two of five clones also express an antigenic determinant of the V beta chain of the T cell receptor (TCR) identified by F23.1 monoclonal antibody. To effectively generate F23.1+ and antigen-specific CTL clones, heterogenous CTL lines were expanded with F23.1 coated Sepharose beads in the presence of TCGF and then stimulated with PR8 virus-infected cells. Thus, both the proliferative activity to PR8 and the expression of the F23.1 marker was increased significantly. Alternatively, F23.1+ T cells were sorted from in vivo primed mice and expanded with PR8 virus-infected stimulator cells in the presence of TCFG. This F23.1+ T cell line exhibited antigen-specific cytotoxicity for PR8 virus-infected target cells. Additionally, in an 'FcR-focused killing' assay only the F23.1+ CTL line and F23.1+ clones lysed Fc receptor bearing target cells in the presence of F23.1 antibody. These findings indicate that antigen-specific and F23.1+ clones can be selected with high efficiency by alternating stimulation with influenza virus-infected cells and with F23.1-coated Sepharose beads or through the use of a cytofluorograph. The usefulness of antigen-specific and F23.1+ CTL clones and other possible strategies for their selection are discussed.