ABSTRACT We designed, synthesized, and identified UIC-94017 (TMC114), a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing a 3( R ),3a( S ),6a( R )- bis -tetrahydrofuranylurethane ( bis -THF) and a sulfonamide isostere which is extremely potent against laboratory HIV-1 strains and primary clinical isolates (50% inhibitory concentration [IC 50 ], ∼0.003 μM; IC 90 , ∼0.009 μM) with minimal cytotoxicity (50% cytotoxic concentration for CD4 + MT-2 cells, 74 μM). UIC-94017 blocked the infectivity and replication of each of HIV-1 NL4-3 variants exposed to and selected for resistance to saquinavir, indinavir, nelfinavir, or ritonavir at concentrations up to 5 μM (IC 50 s, 0.003 to 0.029 μM), although it was less active against HIV-1 NL4-3 variants selected for resistance to amprenavir (IC 50 , 0.22 μM). UIC-94017 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents. Structural analyses revealed that the close contact of UIC-94017 with the main chains of the protease active-site amino acids (Asp-29 and Asp-30) is important for its potency and wide spectrum of activity against multi-PI-resistant HIV-1 variants. Considering the favorable pharmacokinetics of UIC-94017 when administered with ritonavir, the present data warrant that UIC-94017 be further developed as a potential therapeutic agent for the treatment of primary and multi-PI-resistant HIV-1 infections.