We previously demonstrated that proliferation of terminally differentiated Thl clones depends primarily on an interleukin-12 (IL-12)-paracrme mechanism mediated by their interactions with antigen-presenting cells (APC) rather than on an IL-2-autocrine mechanism. Such a Thl clone (4-86, C57BL/6 origin) was cultured with recombinant IL-12 (rIL-12) in the absence of either antigen or APC. Some cells survived for several passages of culture with only rIL-12, and by limiting dilution, several clones highly reactive to rIL-12 alone were obtained. One of these clones, designated 2D6, was found to proliferate strongly in response to less than 1 pg/mL of rIL-12. This clone exhibited the following surface phenotypes: CD3+, T cell receptor (TCR) αβ+, Vβ11+, NK-1.1-; CD4- CD8-; LFA-1+, ICAM-1+; and CD28+, CD80+, CD86+, CTLA-4-. In accordance with high responsiveness to IL-12, 2D6 cells were also found to express IL-12 receptor (IL-12R) as detected by incubation with rIL-12 and then staining with anti-IL-12 monoclonal antibody (mAb). Stimulation of 2D6 with rIL-12 resulted in the expression of interferon-γ (IFN-γ) and IL-10 mRNAs and production of these cytokines. The 2D6 clone responded to IL-2 (vigorously), IL-7 (moderately), and IL-4 (mildly) in addition to IL-12. However, the Ab capture assay using anti-IL-12 mAb enabled us to quantify IL-12-specific activity contained in a given sample. Thus, this study describes the unique features of the IL-12-responsive T cell clone and demonstrates the utilization of this clone in the quantitation of a specific IL-12 activity. J. Leukoc. Biol. 61: 346–352; 1997.