BI 201335 is a hepatitis C virus (HCV) NS3-NS4A (NS3 coexpressed with NS4A) protease inhibitor that has been shown to have potent clinical antiviral activity. It is a highly optimized noncovalent competitive inhibitor of full-length NS3-NS4A proteases of HCV genotypes 1a and 1b with K(i) values of 2.6 and 2.0 nM, respectively. K(i) values of 2 to 230 nM were measured against the NS3-NS4A proteases of HCV genotypes 2 to 6, whereas it was a very weak inhibitor of cathepsin B and showed no measurable inhibition of human leukocyte elastase. BI 201335 was also shown to be a potent inhibitor of HCV RNA replication in vitro with 50% effective concentrations (EC(50)s) of 6.5 and 3.1 nM obtained in genotype 1a and 1b replicon assays. Combinations of BI 201335 with either interferon or ribavirin had additive effects in replicon assays. BI 201335 had good permeability in Caco-2 cell assays and high metabolic stability after incubation with human, rat, monkey, and dog liver microsomes. Its good absorption, distribution, metabolism, and excretion (ADME) profile in vitro, as well as in rat, monkey, and dog, predicted good pharmacokinetics (PK) in humans. Furthermore, drug levels were significantly higher in rat liver than in plasma, suggesting that distribution to the target organ may be especially favorable. BI 201335 is a highly potent and selective NS3-NS4A protease inhibitor with good in vitro and animal ADME properties, consistent with its good human PK profile, and shows great promise as a treatment for HCV infection.