Exposure of the skin to environmental stimuli, such as chemical or physical carcinogens, modifies the local skin environment, including depletion of epidermal Langerhans' cells (LC). Any subsequent exposure of the LC-depleted skin to antigen results in the generation of antigen-specific tolerance. In this study we evaluated the antigen-bearing cells in the draining lymph nodes by capitalizing on the fluorescent nature of the contact sensitizer, fluorescein isothiocyanate (FITC). When FITC was applied to the skin of normal mice, two distinct populations of antigen-bearing cells were identified in the draining lymph nodes. They were classified as either FITC(hi) or FITC(lo) on the basis of their fluorescence intensity and thus the amount of antigen they internalized. Only FITC(lo) cells were detected in the lymph nodes draining FITC-treated murine skin that had been depleted of epidermal LC by prior treatment with the complete carcinogen 9,10-dimethyl 1,2-benzanthracene (DMBA). Functional analysis of these cells revealed that the FITC(hi) cells, but not the FITC(lo) cells, induced antigen-specific T- cell proliferation. Further analysis of the FITC(lo) cells from the DMBA- treated mice demonstrated that these cells had reduced levels of CD80 expression, had substantially reduced levels of CD86 expression and performed poorly as costimulator cells in an anti-CD3-mediated proliferative assay. Nonetheless these cells still induced early signs of T-cell activation and interleukin-12 production. Consequently the FITC(lo) cells migrating from the LC-depleted skin, through a combination of reduced antigen presentation and reduced co-stimulatory activity, induced a state of unresponsiveness or anergy in the responder T cells in a similar manner to that observed when antigen presentation occurs in the absence of costimulation. We propose that these unresponsive, or anergic cells, account for the antigen-specific tolerance observed in these experiments.