In vitro preculture of C3H/He splenocytes for 48 to 96 hr induces aspecific suppressor cells evaluated by their ability to reduce 3H-TdR uptake by fresh syngeneic splenocytes stimulated by different amounts of Concanavalin A (Con A) in vitro. This suppressive activity is obtained in medium containing 10% heat-inactivated fetal calf serum (FCS) or similar amounts of heat-inactivated bovine serum, syngeneic, or allogeneic murine sera but not by unheated FCS. Suppressive activity is resistant to mitomycin C and radiation up to 5,000 R. Exposure of precultured cells to carbonyl iron or plastic adherence, but not to anti-Thy 1.2 serum plus complement, results in removal of the suppressive activity.