Preactivation of ovine peripheral blood and lymph node mononuclear cells with mitogenic doses of concanavalin A (Con A) induced cells which suppressed mitogen-stimulated proliferative responses of untreated autologous and allogeneic responder cells. The degree of suppression varied with preactivating doses of Con A, length of preactivation time, and ratio of preactivated to responder cells. The role of macrophages in generation of suppressor cells was not evaluated. However, macrophages were not required to mediate suppression in cocultures, as lymphoblasts depleted of macrophage by plastic adherence and nylon wool columns mediated equal, and often greater, suppression than unseparated, preactivated cells. Suppressor cell activity in peripheral blood increased from the neonatal period to adulthood. Supernatants from Con A preactivated cell cultures with detectable interleukin-2 activity abrogated suppression when added at 0 and 24 hr of the 72 hr coculture period, suggesting that Con A-induced suppressor cells exert their function by decreasing available levels of IL-2 in the cocultures.