An investigation was made of the properties of cytotoxic T cells induced by Con A and exposure to LCM virus-infected cells. As a basis for such studies, the optimal conditions for in vitro Con A stimulation of in vivo LCM virus-primed C3H mouse splenocytes were determined. The most potent cytotoxicity was obtained when responder cells were cultured in the presence of Con A in a concentration of 2 micrograms/ml for 3 days, but strong cytotoxicity was also measured on days 2 and 4. When stimulation was performed by the homologous antigen maximal response was seen on day 4 although marked cytotoxicity was also noted on day 3. Effector cells produced by the two different procedures showed equally high degrees of cytotoxicity against LCM virus-infected target cells, whereas they did not appear cytotoxic against non-infected targets. If LCM virus-immune mice were treated intravenously with 280 micrograms of Con A per animal, moderate cytotoxicity was demonstrable in splenocytes from these mice 1, 2 and 3 days after treatment. The in vitro generation of secondary cytotoxicity by Con A as well as by the homologous antigen was found to be totally dependent on DNA synthesis. The reactivated cells were investigated for in vivo anti-viral effect by measuring their ability to protect intracerebrally LCM virus-infected mice from a fatal outcome of this infection. LCM virus-primed splenocytes stimulated by the homologous antigen caused complete protection, while Con A-reactivated cells did not protect at all. Secondary cytotoxic cells stimulated by Con A and by LCM virus showed fairly similar in vitro characteristics, but fundamentally different in vivo qualities.