Lopinavir (LPV, formerly ABT-378) is an HIV protease inhibitor (PI) that is co-administered with a small amount of ritonavir (RTV), which greatly increases and sustains the plasma levels of LPV. Lopinavir/ritonavir (LPV/r) has shown potent antiviral activity in both therapy-nai;ve and PI-experienced patients. To assess the effect of pharmacologically relevant ratios of LPV/RTV (LPV/r) on the emergence of resistant HIV in vitro, HIV-1 pNL4-3 was passaged in the presence of increasing concentrations of LPV alone and LPV/r. Passages with fixed 5/1 and 15/1 concentration ratios of LPV/r initially selected I84V and I50V/M46I mutants, respectively. Selection with LPV alone also generated the same initial mutants (I50V/M46I) as the 15/1 LPV/r passage. Further passage produced other mutations previously found to be associated with PI-resistance. Phenotypic susceptibility to both LPV and RTV decreased with successive passages, irrespective of whether RTV was present in the selection experiment. Furthermore, in the two selection experiments that included RTV (at either 5/1 or 15/1 LPV/r ratio), the IC(50) of RTV at each passage evaluated was at least five-fold higher than the concentration of RTV employed at that passage, while the IC(50) of LPV toward the passaged virus was similar to the concentration of LPV used at that passage, indicating that the selective pressure was attributable to LPV and not RTV.