A bacteriophage lambda-based method was used to investigate the development of resistance to protease inhibitors (PI) in one subject infected with human immunodeficiency virus (HIV) type 1 group O who underwent multiple treatment regimens over a period of 4 years. A reduction in the susceptibility to indinavir of 6-fold and a reduction in the susceptibility to saquinavir of 24-fold were recognized after long exposure to these drugs with respect to baseline. The emergence of PI resistance corresponded to the selection of amino acid changes L10V, G48M, F53L, I54V, and L90M at the protease. The results were concordant with those obtained by a drug susceptibility assay with primary HIV isolates.