Two synthetic peptides corresponding to sequences in HIV-1LAI gp41, (aa558-595) and T20 (aa 643-678), are strong inhibitors of HIV-1 viral fusion, having EC50 values of 1 microgram/mL and 1 ng/mL, respectively. Previous work suggested that T21 forms a coiled-coil structure in PBS solution, while T20 is primarily nonhelical, and that the inhibitory action of these peptides occurs after the interaction between the viral gp120 protein and the cellular CD4 receptor [Wild, C.T., Shugars, D. C., Greenwell, T. K., McDanal, C. B., Matthews, T. J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9770 and references therein]. The current study uses sedimentation equilibrium (SE), circular dichroism (CD), and viral-fusion assays to quantitatively investigate peptide structure and peptide-peptide interactions. SE analyses of T21 (1-100 microM) indicate that the peptide self associates via a monomer/dimer/tetramer equilibrium; in addition, T20 is monomeric in the range of 1-10 microM and exhibits a complicated monomer/tetramer equilibrium between 20 and 100 microM. Singular value decomposition analyses of the CD spectra of T21 and T20 indicate that the helical content of these peptides in PBS solution is 90% and 20%, respectively. A structural interaction between the two peptides is detected by CD at several concentration ratios of T20:T21. These experiments emphasize that T20 interacts specifically with the tetrameric form of T21. Truncated forms of T20 also exhibit structural interactions with T21 at varying concentration ratios. The ability of T20 and the truncated peptides to interact structurally with tetrameric T21 correlates with antiviral activity. Implications of these findings are discussed in terms of proposed mechanisms of membrane fusion inhibition and the structural changes which occur in gp41 during membrane fusion.