The discoveries that cyclooxygenase (COX)-2 is an inducible form of COX involved in inflammation and that COX-1 is the major isoform responsible for the production of prostaglandins (PGs) in the gastrointestinal tract have provided a rationale for the development of specific COX-2 inhibitors as a new class of anti-inflammatory agents with improved gastrointestinal tolerability. In the present study, the preclinical pharmacological and biochemical profiles of rofecoxib [Vioxx, also known as MK-0966, 4-(4′-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone], an orally active COX-2 inhibitor, are described. Rofecoxib is a potent inhibitor of the COX-2-dependent production of PGE₂ in human osteosarcoma cells (IC₅₀ = 26 ± 10 nM) and Chinese hamster ovary cells expressing human COX-2 (IC₅₀ = 18 ± 7 nM) with a 1000-fold selectivity for the inhibition of COX-2 compared with the inhibition of COX-1 activity (IC₅₀ > 50 μM in U937 cells and IC₅₀ > 15 μM in Chinese hamster ovary cells expressing human COX-1). Rofecoxib is a time-dependent inhibitor of purified human recombinant COX-2 (IC₅₀ = 0.34 μM) but caused inhibition of purified human COX-1 in a non-time-dependent manner that could only be observed at a very low substrate concentration (IC₅₀ = 26 μM at 0.1 μM arachidonic acid concentration). In an in vitro human whole blood assay, rofecoxib selectively inhibited lipopolysaccharide-induced, COX-2-derived PGE₂ synthesis with an IC₅₀ value of 0.53 ± 0.02 μM compared with an IC₅₀ value of 18.8 ± 0.9 μM for the inhibition of COX-1-derived thromboxane B₂synthesis after blood coagulation. Using the ratio of the COX-1 IC₅₀ values over the COX-2 IC₅₀ values in the human whole blood assay, selectivity ratios for the inhibition of COX-2 of 36, 6.6, 2, 3, and 0.4 were obtained for rofecoxib, celecoxib, meloxicam, diclofenac, and indomethacin, respectively. In several in vivo rodent models, rofecoxib is a potent inhibitor of carrageenan-induced paw edema (ID₅₀ = 1.5 mg/kg), carrageenan-induced paw hyperalgesia (ID₅₀ = 1.0 mg/kg), lipopolysaccharide-induced pyresis (ID₅₀ = 0.24 mg/kg), and adjuvant-induced arthritis (ID₅₀ = 0.74 mg/kg/day). Rofecoxib also has a protective effect on adjuvant-induced destruction of cartilage and bone structures in rats. In a ⁵¹Cr excretion assay for detection of gastrointestinal integrity in either rats or squirrel monkeys, rofecoxib has no effect at doses up to 200 mg/kg/day for 5 days. Rofecoxib is a novel COX-2 inhibitor with a biochemical and pharmacological profile clearly distinct from that of current nonsteroidal anti-inflammatory drugs and represents a new therapeutic class of anti-inflammatory agents for the treatment of the symptoms of osteoarthritis and rheumatoid arthritis with improved gastrointestinal tolerability.