A transframe region within HIV-1 Gag-Pol (referred to as p6*or p6pol), directly linked to the protease (PR) N-terminus, plays a pivotal role in modulating PR activation. To identify specific p6*residues involved in PR activation, we created a series of p6*mutants by making substitutions for conserved p6*residues. Our results indicate that some p6*mutants were defective in terms of virus infectivity, despite displaying a wild-type virus particle processing pattern. Mutations at p6*F8 reduced virus infectivity associated with insufficient virus processing, due in part to impaired PR maturation and RT packaging. Our data strongly suggest that conserved Phe (F) residues at position 8 of p6*are involved in the PR maturation process.