Apratastat is an orally active, potent, and reversible dual inhibitor of tumor necrosis factor-α converting enzyme (TACE) and matrix metalloproteinases (MMPs). This study characterizes the pharmacodynamic (PD) effect of apratastat following oral administration on tumor necrosis factor-alpha (TNF-α) release. Data were obtained from 3 clinical studies carried out in healthy subjects. Apratastat was administered orally in these studies as single doses or multiple doses (twice daily). The inhibition of TNF-α release by apratastat was investigated in studies of in vitro, ex vivo, and in vivo. Inhibitory E(max) models were used to characterize the inhibition of TNF-α release in both in vitro and ex vivo studies. Apratastat inhibited TNF-α release with a population mean IC(50) of 144 ng/mL in vitro and of 81.7 ng/mL ex vivo, respectively. The relationship between TNF-α and apratastat plasma concentration in the endotoxin-challenged study in healthy subjects was well characterized by a mechanism-based PD population model with IC(50) of 126 ng/mL. Apratastat can potently inhibit the release of TNF-α in vitro, ex vivo, and in vivo. Even though the dosage provided adequate exposure to inhibit TNF-α release, apratastat was not efficacious in rheumatoid arthritis (RA). This inconsistency between TNF-α inhibition and the clinical response requires further investigation.