Tumor necrosis factor (TNF) can induce a variety of cellular responses at low picomolar concentrations. This is in apparent conflict with the published dissociation constants for TNF binding to TNF receptors in the order of 100-500 pM. To elucidate the mechanisms underlying the outstanding cellular sensitivity to TNF, we determined the binding characteristics of TNF to both human TNF receptors at 37 degrees C. Calculation of the dissociation constant (Kd) from the association and dissociation rate constants determined at 37 degrees C revealed a remarkable high affinity for TNF binding to the 60-kDa TNF type 1 receptor (TNF-R1; Kd = 1.9 x 10(-11) M) and a significantly lower affinity for the 80-kDa TNF type 2 receptor (TNF-R2; Kd = 4.2 x 10(-10) M). The high affinity determined for TNF-R1 is mainly caused by the marked stability of ligand-receptor complexes in contrast to the transient interaction of soluble TNF with TNF-R2. These data can readily explain the predominant role of TNF-R1 in induction of cellular responses by soluble TNF and suggest the stability of the TNF-TNF receptor complexes as a rationale for their differential signaling capability. In accordance with this reasoning, the lower signaling capability of homotrimeric lymphotoxin, compared with TNF, correlates with a lower stability of the lymphotoxin-TNF-R1 complex at 37 degrees C.