Abstract A 22-amino-acid-long multibranched peptide construct (CLV) derived from the cleavage region (KIEPLGVAPTKAKRR*VVQREKR*) of the human immunodeficiency virus (HIV) type-1 envelope precursor inhibits HIV infection ( Virology,1996, 223, 406–408). We attempted to characterize its activity for Env expressed viaa recombinant vaccinia virus (rVV): gp160 cleavage was delayed, but not impaired, in the presence of CLV (10 μM), whereas neither Env production nor Env membrane expression was significantly altered. Through the synthesis of analogs, we concluded that the presence of a cleavage sequence was required for inhibition of syncytium formation by CLV in rVV-infected CD4 +cell cultures: indeed, a single amino acid residue substitution (R*>S) in the cleavage sites presented by CLV abolished its activity. Other analogs allowed us to further determine the region of CLV which mediates its activity. The ability of a radiolabeled CLV analog to enter cells was also shown. Altogether, these data strongly suggest that CLV acts on Env fusogenicity at least partially through interference with gp160 processing.