BACKGROUND: As it is the very first step of the HIV replication cycle, HIV entry represents an attractive target for the development of new antiviral drugs. In this context, fusion inhibitors are the third class of anti-HIV drugs to be used for treatment, in combination with nucleoside analogues and antiproteases. But the precise mechanism of HIV fusion mechanism is still unclear. Gp41 ectodomain-derived synthetic peptides represent ideal tools for clarifying this mechanism, in order to design more potent anti-HIV drugs. RESULTS: Two soluble trimeric recombinant gp41 proteins, termed Rgp41B and Rgp41A were designed. Both comprise the N- and C-terminal heptad repeat regions of the ectodomain of HIV-1 gp41, connected by a 7-residue hydrophilic linker, in order to mimic the trimeric fusogenic state of the transmembrane glycoprotein. Both recombinant proteins were found to inhibit HIV-1 entry into target cells in a dose-dependent manner. Rgp41A, the most potent inhibitor, was able to inhibit both X4 and R5 isolates into HeLa cells and primary T lymphocytes. X4 viruses were found to be more susceptible than R5 isolates to inhibition by Rgp41A. In order to elucidate how the trimeric recombinant gp41 protein can interfere with HIV-1 entry into target cells, we further investigated its mode of action. Rgp41A was able to bind gp120 but did not induce gp120-gp41 dissociation. Furthermore, this inhibitor could also interfere with a late step of the fusion process, following the mixing of lipids. CONCLUSION: Taken together, our results suggest that Rgp41A can bind to gp120 and also interfere with a late event of the fusion process. Interestingly, Rgp41A can block membrane fusion without preventing lipid mixing. Although further work will be required to fully understand its mode of action, our results already suggest that Rgp41A can interfere with multiple steps of the HIV entry process.