Wild-type and drug-resistant mutated HIV-1 proteases are active as dimers. This work describes the inhibition of their dimerization by a new series of alkyl tripeptides that target the four-stranded antiparallel beta-sheet formed by the interdigitation of the N- and C-monomer ends of each monomer. Analytical ultracentrifugation was used to give experimental evidence of their mode of action that is disruption of the active homodimer with formation of inactive monomer-inhibitor complexes. The minimum length of the alkyl chain needed to inhibit dimerization was established. Sequence variations led to a most potent HIV-PR dimerization inhibitor: palmitoyl-Leu-Glu-Tyr (Kid = 0.3 nM). Insertion of d-amino acids at the first two positions of the peptide moiety increased the inhibitor resistance to proteolysis without abolishing the inhibitory effect. Molecular dynamics simulations of the inhibitor series complexed with wild-type and mutated HIV-PR monomers corroborated the kinetic data. They suggested that the lipopeptide peptide moiety replaces the middle strand in the highly conserved intermolecular four-stranded beta-sheet formed by the peptide termini of each monomer, and the alkyl chain is tightly grasped by the active site groove capped by the beta-hairpin flap in a "superclosed" conformation. These new inhibitors were equally active in vitro against both wild-type and drug-resistant multimutated proteases, and the model suggested that the mutations in the monomer did not interfere with the inhibitor.