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To evaluate protease inhibitor (PI) cross-resistance and reductions in replication capacity conferred by amprenavir-selected mutations.HIV-1IIIB variants derived from passage in increasing concentrations of amprenavir were studied, as well as 3′Gag/protease recombinants derived from them. These strains progressively accumulated mutations at codons 10, 46, 47, 50 and 84 in the protease as well as a p1/p6 cleavage site mutation at codon 449 in Gag. Their susceptibility (IC50) to various PI and their corresponding replication capacities were evaluated by a single-cycle growth assay and compared with measures using competitive cultures and p24 antigen production.Amprenavir susceptibility decreased with increasing numbers of protease mutations. Changes in lopinavir susceptibility paralleled changes in amprenavir susceptibility. Certain amprenavir-selected mutants conferred greater than 10-fold cross-resistance to lopinavir, including PrL10F/M46I/I50V-GagL449F (19-fold) and PrL10F/M46I/I47V/I50V-GagL449F (31-fold). Moreover, one isolate with only two mutations in the protease (L10F/84V) and GagL449F displayed a 7.7-fold increase in lopinavir IC50. Low-level cross-resistance to ritonavir and nelfinavir was also observed. The replication capacity of viruses containing either I84V or I50V was at least 90% lower than the reference virus in the single-cycle assay. The order of relative replication capacity was wild-type > L10F > L10F/I84V > L10F/M46I/I50V > L10F/M46I/I47V/I50V.These results indicate that until more comprehensive genotype–phenotype correlations between amprenavir and lopinavir susceptibility are established, phenotypic testing may be preferable to genotyping to detect cross-resistance, and should be considered when switching patients from a failing amprenavir-containing regimen. This study also provides data on the concordance of replication capacity measurements generated using rapid single-cycle growth and competition assays.