Vigorous CTL response against alloantigens, which is the main effector mechanism in acute allograft rejection, has been well described. Studies to monitor these responses in a quantitative manner has recently taken a new turn following the introduction of new quantitative flow cytometric methods such as USE cell proliferation and intracellular cytokine staining as alternatives to the conventional LDA assays. Although this technique has frequently been used in allogeneic systems in recent years, potential recruitment of non-antigen-specific bystander CD8(+) T cells in the antigen-specific population has not been studied in detail. In addition, the degree of association between the cytotoxicity function and the IFN-gamma expression of CD8(+) T cells has not been elucidated. In this study, we have investigated the bystander recruitment in a mouse allogeneic setting using ex vivo mixed lymphocyte culture (MLC) expanded allogeneic CD8(+) T cells. By using a fluorescent labeling technique, primary unsensitized CD8(+) cells were monitored for their potential to be stimulated to produce IFN-gamma when present in close proximity to activated cells during a 6-h incubation period. In addition, by using two different approaches, direct flow cytometric sorting of IFN-gamma producing cells as well as a direct comparison of IFN-gamma expression and cytolysis in LDA wells, we were able to determine the cytotoxic capacity of IFN-gamma producing CD8(+) T cells. Our results demonstrated that antigen-non-specific CD8(+) T cells are not recruited to produce IFN-gamma in vitro by alloantigen activated T cells. In addition, our results showed only a moderate correlation between the two functions of the alloreactive CD8(+) T cells, and might also suggest the existence of non-cytotoxic subpopulations. (C) 2004 Elsevier B.V. All rights reserved.