Cytotoxic T lymphocytes (CTL) generated in mice against a particular serotype of vesicular stomatitis virus (VSV) were previously shown to cross-reactively lyse syngeneic target cells infected with serologically distinct types of VSV. To analyze the antigenic basis of this T cell cross-reactivity, we generated CTL against VSV-Indiana (VSV-Ind) and established them by limiting dilution as cloned in vitro cell lines. The cells continuously proliferate in medium containing concanavalin A-induced T cell growth factors. All of the cells are Thy-1.2+ and Lyt-2.2+. Lysis by these cells is H-2Dd-restricted, no natural killer cell activity is detectable, and all the clones cross-reactively lyse target cells infected with either VSV-Ind or VSV-New Jersey (VSV-NJ). In addition, no specific blocking of primary, secondary, or cloned anti-VSV CTL was achieved with the use of several monoclonal antibodies specific for the glycoprotein of VSV and capable of neutralizing either VSV-Ind or VSV-NJ. These results suggest that VSV serotype-specific neutralizing antibodies may recognize immunodominant determinants of VSV glycoprotein that are distinct from those recognized by the majority of VSV-specific CTL.