The expression of a previously uncharacterized human hfl-B5 cDNA confers susceptibility for herpes simplex virus (HSV) to porcine cells and fulfills criteria as an HSV entry receptor (A. Perez, Q.-X. Li, P. Perez-Romero, G. DeLassus, S. R. Lopez, S. Sutter, N. McLaren, and A. Oveta Fuller, J. Virol. 79:7419-7430, 2005). Heptad repeats found in the B5 C terminus are predicted to form an alpha-helix for coiled coil structure. We used mutagenesis and synthetic peptides with wild-type and mutant sequences to examine the function of the heptad repeat motif in HSV binding and entry into porcine cells that express B5 and for infection of naturally susceptible human HEp-2 cells. B5 with point mutations predicted to disrupt the putative C-terminal coiled coil failed to mediate HSV binding and entry into porcine cells. Synthetic peptides that contain the single amino acid changes lose the blocking activity of HSV entry. We concluded that the C terminus of B5 contains a functional region that is important for the B5 receptor to mediate events in HSV entry. Structural evidence that this functional region forms coiled coil structures is under investigation. Blocking of HSV interaction with the C-terminal region of the B5 receptor is a new potential target site to intervene in the virus infection of human cells.