The presence, phenotype, and functional characteristics of peripheral blood penicillin-specific T lymphocytes in individuals with cutaneous allergic reactions to penicillin were investigated using in vitro long-term culture techniques. Peripheral blood mononuclear cells from two penicillin-allergic patients were stimulated in vitro with penicillin, and T-cell blasts were clonally expanded by limiting dilution. Seven T-cell clones were derived, all of which were CD3+ CD4 CD8+ HLA-DR+, and produced IL-2 and IFN-γ upon stimulation. T-cell proliferation required the presence of antigen and autologous, but not allogeneic, antigen-presenting cells. In addition to the parent compound, the T-cell clones also developed a proliferative response to penicilloyl, the major metabolite of penicillin. The cloned T-cell lines were found to exhibit marked suppressor activity for Con A mitogenesis. The observed suppressor activity required cell-to-cell contact, as supernatants from these T-cell clones had no comparable inhibitory effect. These findings indicate that there is a predominance of penicillin-specific CD8+ T cells in the peripheral blood of individuals sensitized to beta-lactam antibiotics.