In cells infected with herpes simplex virus 1, intracellular virions in transit along the exocytic pathway carry glycoconjugates that react, in fracture-label technique, with helix pomatia lectin. This lectin is specific for unsubstituted N-acetylgalactosamine, an intermediate sugar added in O-linkage to ser/thr residues in cis-Golgi and then substituted with galactose and sialic acid in the trans-Golgi. Virions in the perinuclear space do not react with helix pomatia lectin. In intracellular transport vesicles and vacules, close to the Golgi complex, virions are positively labeled by helix pomatia lectin and variably labeled by wheat germ agglutinin, a lectin specific for fully mature glycoconjugates. Extracellular virions react only with wheat germ agglutinin. The detection of glycoconjugates at intermediate steps of maturation, coupled with previous results that virions in the perinuclear space carry high mannose oligosaccharides (Torrisi et al., J. Virol. 66, 554-561, 1992), favor the view that maturation of herpes simplex virion envelope proceeds in a stepwise manner along the exocytic pathway. Should transit of virions involve a deenvelopment of enveloped virions followed by reenvelopment of naked nucleocapsids, our results rule out reenvelopment at trans- or post-Golgi compartments and could be consistent with reenvelopment occurring earlier in the exocytic pathway, most likely at the cis-Golgi.