AbstractInterleukin‐12 (IL‐12) is a cytokine that has regulatory effects on T and natural killer (NK) cells and is composed of two disulfide‐bonded subunits, p40 and p35. It was recently reported that supernatants from cultures of mouse IL‐12 (moIL‐12) p40‐transfected COS cells could inhibit IL‐12‐dependent responses in vitro (Mattner, F., et al., Eur. J. Immunol. 1993. 23: 2202). We have further characterized the nature of the inhibitory substance. Purified mouse p40 produced in a baculovirus expression system was found to consist of two species: the p40 monomer and a disulfide‐linked p40 dimer [(p40)2]. The (p40)2 was 25‐ to 50‐fold more active than the p40 monomer in causing specific, dose‐dependent inhibition of IL‐12‐induced mouse concanavalin A (Con A) blast proliferation and could also inhibit IL‐12‐induced interferon‐λ (IFN‐λ) secretion by mouse splenocytes and IL‐12‐dependent activation of mouse NK cells. Competitive binding studies on mouse Con A blasts showed that (p40)2 was equally effective as moIL‐12 in competing with 125I‐labeled moIL‐12 ([125I]moIL‐12) for binding to mouse Con A blasts. However, in contrast to moIL‐12, mouse (p40)2 displayed little ability to compete with 125I‐labeled human IL‐12 (huIL‐12) for binding to high‐affinity IL‐12 receptors (IL‐12R) on human phytohemagglutinin (PHA) blasts and caused little or no inhibition of huIL‐12‐induced human PHA blast proliferation. Nonetheless, mouse (p40)2 was equally effective as moIL‐12 in competing with [125I] huIL‐12 for binding to COS cells transfected with the human IL‐12R β subunit and expressing low‐affinity IL‐12 binding sites. These results suggest that (i) the majority of the structural determinants required for binding of IL‐12 to its receptor are contained within the p40 subunit, but p35 is required for signaling, (ii) the p40 subunit of IL‐12 interacts with the β subunit of IL‐12R, and (iii) (p40)2 may be a suitable IL‐12 antagonist for studying the role of IL‐12 in various immune responses in vivo as well as in vitro. Further studies are required to determine whether or not (p40)2 is produced by normal lymphoid cells and is a physiologic regulator of IL‐12 activity.