Twenty-four of over 24,000 patients genotyped over the past 3 years were found to have human immunodeficiency virus (HIV) isolates that possess an insert in the protease gene. In this report, we evaluated the spectrum of protease gene insertion mutations in patient isolates and analyzed the effect of these various insertion mutations on viral phenotypes. The inserts were composed of 1, 2, 5, or 6 amino acids that mapped at or between codons 35 and 38, 17 and 18, 21 and 25, or 95 and 96. Reduced susceptibility to protease inhibitors was found in isolates which possess previously reported drug resistance mutations. Fitness assays, including replication and competition experiments, showed that most of the isolates with inserts grew somewhat better than their counterparts with a deletion of the insert. These experiments demonstrate that, rarely, insertion mutations can develop in the HIV type 1 protease gene, are no more resistant than any other sequences which have similar associated resistance mutations, and can provide a borderline advantage in replication.