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The redistribution of mutations related to protease inhibitor (PI) resistance after a PI-sparing regimen in human immunodeficiency virus (HIV)-infected, highly PI-experienced patients was prospectively assessed. Twenty-five patients failing a PI-including regimen were given PI-sparing antiretroviral therapy, and then followed for 24 weeks after PI resumption. Genotyping was performed by direct sequencing before and during the PI-sparing regimen. The median (interquartile range, IQR) baseline CD4⁺ T-lymphocyte count was 198 (120–255) cells/µl, and the median HIV-RNA level was 82,000 (41,000–300,000) copies/ml. Patients had experienced a median of 4.5 (4–5.25) PIs. The median number of PI mutations was eight (6–9). The PI-sparing regimen consisted of a median of three (3–4) drugs and lasted for a median of 53 (24–67) weeks. At the end of the study, the median number of PI mutations was 6.5 (6–9). The median change in the number of PI mutations was −1 (IQR from −1 to 0). A reduction from baseline was observed in 13 cases (52%); nine (36%) showed no change and three (12%) showed an increased number of PI substitutions. In highly PI-experienced patients, a PI-sparing regimen may lead to a reduction, no change, or increase in the number of PI mutations. The reduction is negligible in most cases.