Previous use of the HIV-1 protease inhibitor saquinavir resulted in the infrequent appearance of mutations in the HIV-1 protease gene associated with resistance. We have examined the ability of saquinavir to select for resistance mutations. In multiple selections of HIV-1 in cell culture with saquinavir, similar patterns of mutations were reproducibly observed and the number of mutations increased with increasing selective pressure. In a small number of subjects who showed an antiviral response when saquinavir was added to their therapeutic regimen, similar mutations were detected in viral genomic RNA in vivo after 30 to 40 weeks of therapy. These results indicate that saquinavir can select for resistance mutations and suggest that the infrequent appearance of these mutations in vivo is the result of low drug exposure. These results also predict that the use of higher levels of saquinavir will lead to an even greater frequency of resistance mutations in patients who fail therapy.