The establishment and characterization of cloned natural suppressor (NS) cell lines derived from the spleen of neonatal BALB/c mice are described. Cloned NS cells suppress the mixed leukocyte reaction (MLR) between normal adult responder and stimulator spleen cells with a 50-fold greater efficiency than fresh neonatal cells. Suppressive activity of both cells did not depend on the haplotype of the responder or stimulator cells, and was radioresistant. Cloned NS cells did not inhibit the uptake of [3H]thymidine by HT-2 cells proliferating in response to interleukin 2 (IL-2), nor the in vitro secretion of IL-1 by macrophages in response to lipopolysaccharide. Several experiments indicated that absorption of IL-2 could not explain the suppression of the MLR by the NS cells in the range of cell numbers tested. The results suggest that NS cells may suppress the MLR by interfering with early stages of T cell activation. The cell surface of a cloned NS cell line was examined using immunofluorescence staining, and was strongly positive for the Thy-1.2, Ly-5, and asialo-GM1 antigens. However, Lyt-1, Lyt-2, surface Ig, IE, MAC-1, and Fc and C3 receptor markers were not detected. In addition, NS cells showed no cytolytic activity against the YAC-1 target cell line. On the basis of these findings, cloned NS cells do not appear to be mature T cells, B cells, macrophages, or NK cells. The development of cloned NS cells may be useful in determining the identity and mechanism of action of nonspecific suppressor cells in the neonatal spleen, and their role in neonatal tolerance and maternal-fetal relationships.