The kinetics of sensitization against H2-antigens leading to cytotoxicity were studied in a mouse allogeneic in vitro system, where the antigen-reactive lymphocytes can be separated at any precise moment from the allogeneic sensitizing fibroblasts and subcultured isogenetically. To achieve optimal sensitization 48 h of close contact with antigenic fibroblasts is necessary. At this time no cytotoxicity is detectable. The sensitization period of 48 h is followed by a differentiation period on days 3–4, which seems to be characterized by an enhanced stickiness of the sensitized lymphocytes to the allogeneic feeder layer. Five days of culture are needed to develop maximum cytotoxicity.