We examined the prevalence of cleavage site mutations, both within and outside the gag region, in 28 protease inhibitor (PI) cross-resistant patients treated with indinavir, ritonavir, and/or saquinavir compared to control patients treated with reverse transcriptase inhibitors. Three human immunodeficiency virus protease cleavage sites within gag (p2/NC, NC/p1, and NC/TFP) showed considerable in vivo evolution before and after therapy with indinavir, ritonavir, and/or saquinavir. Another gag cleavage site (p1/p6(gag)) showed a trend compared to matched controls. The other eight recognized cleavage sites showed relatively little difference between PI-resistant cases and controls. An A-->V substitution at the P2 position of the NC/p1 and NC/TFP cleavage sites was the most common (29%) change selected by the PIs used in this study.