Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) results from mutations in the viral protease (PR) that reduce PI binding but also decrease viral replicative capacity (RC). Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6) with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists.