The virion of herpes simplex virus type 1 (HSV-1) consists of three structural components which include the envelope, tegument, and capsid. Little is known about the organization and potential interaction of proteins of the tegument and envelope. In this report, studies with chemical cross-linking reagents were initiated to determine the arrangement of the HSV-1 glycoprotein B (gB) on the viral envelope and its interactions with other virion proteins. With use of four homobifunctional N-hydroxysuccinimide ester reagents, gB was cross-linked to dimers and eventually high molecular weight complexes. In addition, four virion structural proteins were detected to be associated with gB as well as gD and gH but not with gC when cross-linked with dithiobis(succinimidyl propionate). Experimental results indicated that these four proteins are likely to represent viral tegument proteins and one of these proteins was immunologically identified as the known tegument protein, VP16.