The effect of D-penicillamine on in vitro mitogen-triggered human peripheral blood lymphocyte DNA synthesis was examined. D-penicillamine caused a modest degree of inhibition of responsiveness. Since D-penicillamine avidly chelates copper, a possible additive effect of D-penicillamine and copper salts on mitogen-induced lymphocyte proliferation was investigated. The addition of various copper salts to D-penicillamine-containing cultures resulted in a marked augmentation in the degree of inhibition observed. Specificity for the synergy between D-penicillamine and copper was suggested by the failure of iron, zinc, or gold salts to augment the inhibition produced by D-penicillamine alone. However, a number of other thiols, but not disulfides, could substitute for D-penicillamine in inhibiting mitogen responsiveness in the presence of copper salts. Preincubation of peripheral blood mononuclear cells with D-penicillamine and CuSO4 resulted in diminished mitogen responsiveness on subsequent culture in the absence of the inhibitors. Data are presented to indicate that preincubation with D-penicillamine and CuSO4 directly affected the ability of the T lymphocytes to respond to mitogens but did not alter the capacity of the monocytes to act as accessory cells in this response.