Human immunodeficiency virus type 1 (HIV‐1) Vif protein is required for productive HIV‐1 infection of peripheral blood lymphocytes and macrophages in cell culture and for pathogenesis in the SCID‐hu mouse model of HIV‐1 infection. Vif inhibits the viral protease (PR)‐dependent autoprocessing of truncated HIV‐1 Gag‐Pol precursors expressed in bacterial cells and efficiently inhibits the PR‐mediated hydrolysis of peptides in cell‐free systems. The obstructive activity of Vif has been assigned to the 92 amino acids residing at its N′‐terminus (N‐Vif). To determine the minimal Vif sequence required to inhibit PR, we synthesized overlapping peptides derived from N‐Vif. These peptides were then assessed, using two in vitro and two in vivo systems: (i) inhibition of purified PR, (ii) binding of PR, (iii) inhibition of the autoprocessing of the Gag‐Pol polyprotein expressed by a vaccinia virus vector, and (iv) inhibition of mature virus production in human cells. The peptides derived from two regions of N‐Vif encompassing residues Tyr‐30–Val‐65 and Asp‐78–Val‐98, inhibited PR activity in both the in vitro and the in vivo assays. Thus, these peptides can be used as lead compounds to design new PR inhibitors.