Tolerance and sensitivity to a potent contact sensitizer were studied in vivo and in vitro. Contact hypersensitivity to 2,4-dinitro-1-fluorobenzene (DNFB) was induced in BALB/c mice by two daily paintings with 0.5% DNFB. The degree of hypersensitivity was assayed 4 days after the second painting by measuring the increments of ear thickness 24 hr after challenging the ears with 0.2% DNFB. Ear swelling was maximum at 24 hr and the histology was compatible with delayed type hypersensitivity. Specific tolerance to DNFB was produced by a single i.v. injection of 15 mg of 2,4-dinitrobenzene-1-sulfonic acid sodium salt (DNB·SO3Na) 7 days prior to sensitization. DNB SO3Na given 2 days before, the same day as or 1 or 3 days after sensitization resulted in only partial unresponsiveness. Tolerance and sensitization were also measured by in vitro stimulation of DNA synthesis in cultured lymphoid cells. Lymph node cells taken 5 days after sensitization were specifically stimulated by DNP-BSA (but not by BSA) in vitro as assayed by increase of 3H-thymidine uptake. This response was dependent on the presence of T cells. Cells from non-sensitized mice responded poorly to DNP-BSA stimulation. Lymph node cells from tolerant mice responded 71 to 87% less than did sensitized cells. The results are discussed in terms of possible mechanisms of tolerance. In the context of this model of contact sensitivity, this appears to be tolerance at the T-cell level.