We have previously shown that the first generation human immunodeficiency virus (HIV) fusion inhibitor T20 (Fuzeon) contains a critical lipid-binding domain (LBD), whereas C34, another anti-HIV peptide derived from the gp41 C-terminal heptad repeat, consists of an important pocket-binding domain (PBD), and both share a common 4-3 heptad repeat (HR) sequence (Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612-9620). T1249, the second generation HIV fusion inhibitor, has both LBD and PBD but a different HR sequence, suggesting that these three anti-HIV peptides may have distinct mechanisms of action. Here we rationally designed a set of peptides that contain multiple copies of a predicted HR sequence (5HR) or the HR sequence plus either LBD (4HR-LBD) or PBD (PBD-4HR) or both (PBD-3HR-LBD), and we compared their anti-HIV-1 activity and biophysical properties. We found that the peptide 5HR exhibited low-to-moderate inhibitory activity on HIV-1-mediated cell-cell fusion, whereas addition of LBD and/or PBD to the HR sequence resulted in a significant increase of the anti-HIV-1 activity. The peptides containing PBD, including PBD-4HR and PBD-3HR-LBD, could form a stable six-helix bundle with the N-peptide N46 and effectively blocked the gp41 core formation, whereas the peptides containing LBD, e.g. 4HR-LBD and PBD-3HR-LBD, could interact with the lipid vehicles. These results suggest that the HR sequence in these anti-HIV peptides acts as a structure domain and is responsible for its interaction with the HR sequence in N-terminal heptad repeat, whereas PBD and LBD are critical for interactions with their corresponding targets. T20, C34, and T1249 may function like 4HR-LBD, PBD-4HR, and PBD-3HR-LBD, respectively, to interact with different target sites for inhibiting HIV fusion and entry. Therefore, this study provides important information for understanding the mechanisms of action of the peptic HIV-1 fusion inhibitors and for rational design of novel antiviral peptides against HIV and other viruses with class I fusion proteins.