Mumps virus-specific cytotoxic T lymphocytes (CTL) were induced by incubating peripheral lymphocytes from healthy blood donors with autologous mumps virus-infected stimulator cells. Maximum cytotoxicity was generated after 5 to 7 days in culture. No cytotoxic activity was induced in the absence of viral antigens. The cytotoxicity induced in vitro was a secondary immune response, because no specific lytic activity could be generated in lymphocyte cultures from mumps seronegative donors. The effector cells were restricted by the HLA complex in a "hierarchical" pattern. The major restriction antigens for secondary CTL were certain HLA B determinants such as B18, B27, Bw25, Bw62, and Bw63. Mumps responses in spite of high [3H]-thymidine uptake in sensitized cultures. The present data suggest that mumps virus-specific CTL may be directly regulated by certain genes of the HLA B locus.