Human mixed lymphocyte cultures (MLC) have been observed to generate cytotoxicity not only against allospecific targets but also against autochthonous control target cells. This work investigates the types of target cells susceptible to the MLC-activated effectors and also analyzes the time course of the generation of these activities. Normal human peripheral blood lymphocytes (PBL) were sensitized to normal allogeneic mitomycin C-treated PBL in MLC and subsequently were tested for cytotoxicity against various targets in a 51Cr-release cell-mediated lympholysis (CML) assay. Two types of cytotoxic activities were clearly distinguishable by the types of target cells susceptible to the effectors: a) The allocytotoxicity was directed at the relevant allogeneic Con A blasts (alloblasts), but not to the autochthonous control Con A blast (autoblast) targets. b) In contrast, the “anomalous” cytotoxicity, which was originally defined as alloactivated cytotoxicity against the autochthonous lymphoblastoid cell line (auto-LCL) target, was shown to have a broad range of cytotoxic activity against all cultured cell line targets tested. The allocytotoxicity was further distinguished from the “anomalous” reactivity by studies on the time of development in culture of these activities. The peak anomalous activity always occurred 1 or 2 days earlier and declined more rapidly than the peak allocytotoxicity. Experiments to find common antigenic determinants between the sensitizing cells and targets susceptible to anomalous cytotoxicity revealed no obvious antigenic relationships.