The immunoregulatory effect of non-transferrin-bound iron (Fe3+) on the proliferative and cytotoxic responses of normal human T lymphocytes was studied using a sensitive limit-dilution technique capable of detecting the responses of individual lymphocytes. Iron, present in the form of ferric citrate at concentrations from 0.03 to 1.0 mmol/L, significantly reduced the cloning frequency of peripheral blood T lymphocytes. The effect of iron appeared, however, to be targeted to individual clones in that some clones that did grow in the presence of iron achieved a normal rate of proliferation. Thus, iron was not non-specifically toxic. At these same concentrations ferric citrate also produced significant reductions in the cloning frequency of CD4+ CD8-precursor T lymphocytes. Reductions in the response of T lymphocyte precursors capable of cytotoxic activity occurred in the presence of ferric citrate from 0.1 to 1.0 mmol/L. These data support the hypothesis that non-transferrin-bound iron has an immunoregulatory role in cell-mediated immunity.