To characterize the T cells that are activated during the induction of contact hypersensitivity (CH), two sets of studies were conducted: 1) dinitrophenol (DNP)-specific proliferative responses of T cells in draining lymph nodes of BALB/c mice sensitized epicutaneously to dinitrofluorobenzene (DNFB) were examined, and 2) from these lymph node cells, DNP-specific T cells were cloned by limiting dilution microculture and analyzed by FACS for surface markers, by RT-PCR, HT2 bioassay and ELISA for cytokine expression at mRNA and protein levels respectively, and by proliferation assay for cytokine and antigen-presenting cell (APC) requirements. Our results show that alpha beta TCR-bearing T cells of both the CD4+ and CD8+ subtypes from lymph nodes of DNFB-skin-painted mice proliferate specifically to dinitrobenzene sulfonate (DNBS) in vitro. Four DNP-specific, CD4+ T-cell clones were characterized: clone 5S4 secreted IL-4 and required Il-4 for optimal growth; clone 5S10 secreted IL-2 and required IL-2 for optimal growth; clone 5S2 secreted IL-4 but required IL-2 for optimal growth; and clone 5S8 secreted IL-2 predominantly at 5 months, but switched to production of IL-4 at 7 months. All four clones secreted IL-10, and proliferated to DNBS when Langerhans cell (LC)-enriched epidermal cells were used as APC. These findings indicate that heterogeneous populations of DNP-specific T cells are activated in draining lymph nodes during the induction of CH to DNFB in BALB/c mice.