Increased susceptibility to the protease inhibitors saquinavir and amprenavir has been observed in human immunodeficiency virus type 1 (HIV-1) with specific mutations in protease (V82T and N88S). Increased susceptibility to ritonavir has also been described in some viruses from antiretroviral agent-naïve patients with primary HIV-1 infection in association with combinations of amino acid changes at polymorphic sites in the protease. Many of the viruses displaying increased susceptibility to protease inhibitors also had low replication capacity. In this retrospective study, we analyze the drug susceptibility phenotype and the replication capacity of virus isolates obtained at the peaks of viremia during five consecutive structured treatment interruptions in 12 chronically HIV-1-infected patients. Ten out of 12 patients had at least one sample with protease inhibitor hypersusceptibility (change ≤0.4-fold) to one or more protease inhibitor. Hypersusceptibility to different protease inhibitors was observed at variable frequency, ranging from 38% to amprenavir to 11% to nelfinavir. Pairwise comparisons between susceptibilities for the protease inhibitors showed a consistent correlation among all pairs. There was also a significant relationship between susceptibility to protease inhibitors and replication capacity in all patients. Replication capacity remained stable over the course of repetitive cycles of structured treatment interruptions. We could find no association between in vitro replication capacity and in vivo plasma viral load doubling time and CD4+ and CD8+ T-cell counts at each treatment interruption. Several mutations were associated with hypersusceptibility to each protease inhibitor in a univariate analysis. This study extends the association between hypersusceptibility to protease inhibitors and low replication capacity to virus isolated from chronically infected patients and highlights the complexity of determining the genetic basis of this phenomenon. The potential clinical relevance of protease inhibitor hypersusceptibility and low replication capacity to virologic response to protease inhibitor-based therapies deserves to be investigated further. Copyright © 2005, American Society for Microbiology. All Rights Reserved.