A panel of monoclonal antibodies (MAb) reactive with the vesicular stomatitis virus (VSV) glycoprotein (G-protein) was used to inhibit lysis of infected target cells by cytotoxic T lymphocytes (CTL) reactive with VSV serotypes Indiana and New Jersey (VSV-Ind, VSV-NJ). MAb to distinct epitopes on the VSV-NJ G-protein were able to block lysis of VSV-NJ-infected targets using anti-VSV-NJ CTL. Furthermore, when a combination of MAb to three distinct epitopes was used, increased inhibition was observed when compared to single epitope blocking. Cross-reactive CTL generated using either VSV-NJ or VSV-Ind could be inhibited by MAb specific for the heterologous virus in blocking assays. Cross-reactive MAb also were able to block both serotype-specific and cross-reactive lysis. Antigenic variants selected with some of the MAb used in blocking studies were used to infect target cells. Reduction in lysis was not observed using variants with mutations at several epitopes. This result suggests that blocking by MAb involves steric hindrance of CTL-recognized determinants proximal to the MAb binding site.