The core complex is a structure involved in the fusion mechanism of many viruses, as well as in intracellular vesicle fusion. A powerful approach for studying the dynamic stages of HIV-1-cell fusion utilizes DP178, a core complex inhibitory peptide derived from the known sequence of the virus. Strikingly, we show that fatty acids can replace the entire C-terminal region of DP178, known to play a crucial role in the activity of the peptide. The inhibitory activity correlated with the length of the fatty acid, with the direction of fatty acid attachment (N- or C-terminus) and, as envisioned by a new triple staining assay, with the concentration of the peptides on cells. Our findings indicate, for the first time, the C-terminal boundary of the endogenous core structure in situ and establish that the C-terminal region of DP178 functions mainly as an anchor to the cell membrane. Apart from the mechanistic implications, such short lipopeptides provide new, promising fusion inhibitors. Because the fusion mechanism of HIV-1 is shared by other pathogen-enveloped viruses and by intracellular vesicle fusion, our results might influence the research and therapeutic efforts in these systems as well.--Wexler-Cohen, Y. and Shai, Y. Demonstrating the C-terminal boundary of the HIV 1 fusion conformation in a dynamic ongoing fusion process and implication for fusion inhibition.