To obtain further insight into the informatory possibilities of the TakasugiKlein microcytotoxicity test (MCT), mouse spleen cells were prepared after a single immunization with 5 × 107 H-2 incompatible cells and tested for reactivity against mouse fibroblasts. The following three aspects were investigated: (1) Specificity of the reaction: various mixtures of different target cells revealed absence of innocent bystander kill even after 48 h test incubation. (2) Kinetics of the reactivity after short-term (20 h) or long-term (48 h) incubation: the former disappeared rapidly, whereas the latter remained detectable beyond day 60 after immunization. This suggests the activity of different cells: an early killer cell and a later increasingly dominating effector cell which requires long-term incubation with the target. (3) Characterization of the effector cells: anti-theta-serum treatment abolished MCT reactivity completely at early and late stages after immunization. Nylon wool effluent (T-enriched) spleen cells were absolutely more active after short-term but became relatively less effective after long-term incubation as compared to nonseparated spleen cells, suggesting a higher adhesiveness of the cells which operate the 48 h reaction. The findings may demonstrate the usefulness of the MCT for in vitro studies on presence and behavior of different cells involved in allograft immunity in vivo.