Paired immunoglobulin (Ig)-like type 2 receptor alpha (PILRalpha) and PILRbeta are paired receptors that are highly homologous to each other. When engaged by ligand, PILRalpha is inhibitory whereas PILRbeta is activating. PILRalpha is a newly identified herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) receptor and is associated with membrane fusion and entry activity of HSV-1. PILRalpha is a 303-amino-acid protein with an Ig-like V (variable)-type domain from amino acid 31 to 150, whereas PILRbeta is a 217-amino-acid protein with an Ig-like V-type domain from amino acid 21 to 143. We report that PILRbeta is not a receptor for HSV-1 and HSV-2. Domain swaps between PILRalpha and PILRbeta reveal that the Ig-like V-type domain of PILRalpha, but not PILRbeta, plays a critical role in cell membrane fusion activity and the binding of PILRalpha to gB. Individual replacement of 13 amino acids in PILRalpha showed that most of these mutations had no effect on cell fusion activity. However, mutation of the tryptophan residue at amino acid 139 significantly impaired cell fusion activity for HSV-1 and eliminated binding to gB.