Apolipoprotein A‐I (apoA‐I), the major protein component of serum high‐density lipoproteins (HDL), was found to inhibit herpes simplex virus (HSV)‐induced cell fusion at physiological (∼ 1 μM) concentrations, whereas HDL did not exert any inhibitory effect. Lipid‐associating, synthetic amphipathic peptides corresponding to residues 1–33 (apoA‐I[1–33]) or residues 66–120 (apoA‐I[66–120]) of apoA‐I, also inhibited HSV‐induced cell fusion, whereas a peptide corresponding to residues 8–33 of apoA‐I (apoA‐I[8–33]), which fails to associate with lipids, did not exert any inhibitory effect. These results suggest that lipid binding may be a prerequisite for peptide‐mediated fusion inhibition. Consistent with this idea, a series of lipid‐binding 22‐amino‐acid‐residue‐long synthetic amphipathic peptides that correspond to the amphipathic helical domains of apoA‐I (A‐I consensus series), or 18‐residue‐long model amphipathic peptides (18A series), were found to exert variable levels of fusion‐inhibitory activity. The extent of fusion‐inhibitory activity did not correlate with hydrophobic moment, hydrophobicity of the nonpolar face, helix‐forming ability, or lipid affinity of the different peptides. Peptides in which the nonpolar face was not interrupted by a charged residue displayed greater fusion‐inhibitory activity. Also, the presence of positively charged residues at the polar‐nonpolar interface was found to correlate with higher fusion‐inhibitory activity. Copyright © 1991 Wiley‐Liss, Inc.