The novel virucidal protein cyanovirin-N (CV-N) binds with equally high affinity to soluble forms of either H9 cell-produced or recombinant glycosylated HIV-1 gp120 (sgp120) or gp160 (sgp160). Fluorescence polarization studies showed that CV-N is also capable of binding to the glycosylated ectodomain of the HIV-envelope protein gp41 (sgp41) (as well as SIV glycoprotein 32), albeit with considerably lower affinity than the sgp120/CV-N interaction. Pretreatment of CV-N with either sgp120 or sgp41 abrogated the neutralizing activity of CV-N against intact, infectious HIV-1 virions. Isothermal calorimetry and optical biosensor binding studies showed that CV-N bound to recombinant sgp120 with a K(d) value ranging from 2 to 45 nM and to sgp41 with a K(d) value of 606 nM; furthermore, they indicated an approximate 5:1 stoichiometry for CV-N binding to sgp120 and a 1:1 stoichiometry for CV-N binding to sgp41. Circular dichroism studies additionally illuminated the binding of CV-N with both sgp120 and sgp41, providing the first direct evidence that conformational changes are a consequence of CV-N interactions with both HIV-1 envelope glycoproteins.