The predicted amino acid sequence of herpes simplex virus type 1 glycoprotein C (HSV-1 gC) shows that it has the features of a typical type 1 integral membrane protein: a cleavable N-terminal signal sequence, a glycosylated ectodomain, a single transmembrane domain, and a small, charged cytoplasmic domain. In an earlier investigation of the function of the gC cytoplasmic domain, it was shown that the gC synthesized by a gC mutant, dl2, which lacked the last three residues of the transmembrane domain and the entire cytoplasmic domain, was initially synthesized as a membrane bound glycoprotein, but was not stably anchored in the plasma membrane. In this study, we generated a panel of four HSV-1 gC mutants with novel cytoplasmic domains in order to further delineate the role of this domain in stable anchoring and to investigate the role of charged residues in this process. The cytoplasmic domain mutants produced significant quantities of a novel precursor (pgC-86K), approximately 6K smaller than the wild-type gC precursor. The quantity of pgC-86K correlated with the number of positive charges in the cytoplasmic domain. Although the nature of the novel form of the precursor is unclear, its correlation with cytoplasmic domain charge suggests that an important function of this domain is to influence gC processing. Significantly, restoration of the carboxy-terminal 3 amino acids of the gC transmembrane domain restored the wild-type anchoring phenotype to gC, indicating that the cytoplasmic domain is not required for membrane anchoring. Further characterization of dl2 gC confirmed that this glycoprotein is not released from the membrane by proteolysis. We suggest that the addition of three additional hydrophobic residues to the dl2 transmembrane domain increases its hydrophobicity enough to stabilize membrane anchoring of the glycoprotein.