Nonspecific cytotoxic T-cell populations, derived from murine fetal calf serum (FCS)-precultured cells expanded in interleukin 2 (IL-2)-containing supernatant (operationally defined as FCS-CM-expanded cells), were investigated for their inactivating properties on syngeneic lymphoid cell populations containing alloreactive cytotoxic-T-lymphocyte precursors (CTL-P). CTL-P were detected and quantitated in a limiting dilution mixed leukocyte microculture (micro-MLC) system supplemented with IL-2. The data show a dramatic decrease in relevant CTL-P frequency in populations of fresh or Day 2 in vitro-alloactivated peripheral blood leukocytes (PBL) after coculturing them for 24 hr with syngeneic mitomycin C-treated populations of FCS-CM-expanded cells. On the contrary, no decrease in CTL-P frequency was observed when Day 7, instead of fresh or Day 2, in vitro-alloactivated PBL were used as responding cells. Throughout these experiments, it was clearly shown that a decrease or an absence of CTL response in the micro-MLC was neither due to a lack of IL-2 nor to a premature destruction of the stimulating cells by the inhibiting FCS-CM-expanded cells still present in the culture. FCS-CM-expanded cells can destroy (in a 3-hr 51Cr-release assay) Day 2 alloreactive PBL populations, and this raises the possibility that the direct inactivation of CTL-P by FCS-CM-expanded cells could result from their cytolytic activities.