The role of a cytokine synthesis inhibitory factor, interleukin-10 (IL-10), in the induction and maintenance of neonatal transplantation tolerance was studied in mice. We showed that neonatal spleen cells (NSC) significantly inhibited interleukin-2 (IL-2) production by activated T cells from adult mice. Simultaneously we demonstrated a high expression of the IL-10 gene in stimulated spleen cells from newborn mice. However, neutralizing monoclonal antibody (mAb) anti-IL-10 did not abolish the NSC-mediated suppression of IL-2 production. IL-10, therefore, does not appear to be the principal inhibitory molecule responsible for the suppression of IL-2 production. Similarly, specific alloantigen-activated spleen cells from adult tolerant animals were profoundly hyporeactive in IL-2 production. This hyporeactivity was not reversed to a positive reactivity in the presence of mAb anti-IL-10. In addition, anti-IL-10 antibody enhanced proliferation in mixed lymphocyte cultures of cells from both control and tolerant animals, but the antibody did not abrogate specific hyporeactivity of cells from tolerant mice. These results thus showed that newborn animals were nonspecifically and tolerant animals specifically deficient in IL-2 production, but that IL-10 in neither case appeared to be responsible for this IL-2 hyporeactivity.