Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by ␣-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.Herpes simplex virus (HSV) is a neurotropic agent responsible for episodic cold sores and genital legions. After primary infection of mucosal epithelial cells, the virus establishes lifelong latency in sensory neurons, from which it periodically reactivates. After reactivation, the virus migrates along the axons and infects cells at the site of primary infection, causing painful blisters on the surface of the lips in the case of HSV type 1 (HSV-1) or of the genital mucosa for the closely related HSV-2 (48).A critical event in the life cycle resides in the entry of the virus into target cells. Recent progress has been made in understanding the mechanism governing this process (reviewed in references 21 and 38). Of the 12 different glycoproteins of the viral envelope, 4 have essential functions for entry, namely, glycoprotein B (gB), gD, gH, and gL. First, the virion attaches by interaction of gC and gB with cell surface heparan sulfate proteoglycans (42). Although not essential for entry, this step provides stable interactions between the virion and the cell that favor the next steps. These include the association of gD with one of its three identified receptors-HVEM, nectin-1, and 3-O-sulfated...