The effect was studied of D-penicillamine (D-PAm) and other thiol compounds (concentration range, approximately 10(-4)-3.4 X 10(-3) M) on mouse spleen cells, stimulated in culture by concanavalin A or by mixed lymphocyte interaction. Thiols initially enhanced the rate of incorporation of [3H] thymidine into DNA of these cells. The enhanecment was typically followed by a sharp decline in activity to a level often below that of thiol-free cultures. The time which elapsed between adding the thiol and the decline in activity depended on the concentration of the particular thiol used. Exceptional among thiols studied was L-cysteine, which lacked inhibitory properties. Variation of the L-cystine content of the medium used here influenced the effect of thiols such as D-PAm on the cells, an increase in cystine content favouring the retention of high activity. It was found that D-PAm reacted slowly with a constituent of the culture medium, causing the latter eventually to lose its capacity to maintain cells. The effects of this reaction could be counteracted by adding L-cystine, L-cysteine, and certain other thiol compounds. It was concluded that DPAm combined with L-cystine in the medium to form the mixed disulphide, which evidently could not be utilized by the cells as a source of L-cystine. Deprivation of this essential nutrient accounted for the eventual inhibition of [3H] thymidine incorporation in cultures exposed to thiols. The initial, enhancing effect of thiols on stimulated lymphocytes represented a modulation of the response to the primary stimulant: there was no evidence that thiols were themselves mitogenic.