Substitution of leucine for isoleucine at residue 50 (I50L) of human immunodeficiency virus (HIV) protease is the signature substitution for atazanavir (ATV) resistance. A unique phenotypic profile has been associated with viruses containing the I50L substitution, which produces ATV-specific resistance and increased susceptibility to most other approved HIV protease inhibitors (PIs). The basis for this unique phenotype has not been clearly elucidated. In this report, a direct effect of I50L on the susceptibility to the PI class is described. Cell-based protease assays using wild-type and PI-resistant proteases from laboratory and clinical isolates and in vitro antiviral assays were used to demonstrate a strong concordance between changes in PI susceptibility at the level of protease inhibition and changes in susceptibility observed at the level of virus infection. The results show that the induction of ATV resistance and increased susceptibility to other PIs by the I50L substitution is likely determined at the level of protease inhibition. Moreover, the I50L substitution functions to increase PI susceptibility even in the presence of other primary and secondary PI resistance substitutions. These findings may have implications regarding the optimal sequencing of PI therapies necessary to preserve PI treatment options of patients with ATV-resistant HIV infections.