The UL16 gene of herpes simplex virus maps within the intron of the UL 15 gene. This report shows the following: (i) A polyclonal antiserum directed against a bacterial fusion protein containing glutathione S-transferase fused to the C-terminus of the UL 16 gene reacted with an apparent M(r) 40,000 protein in HSV-1 infected cell lysates. (ii) The protein encoded by UL 16 was dependent on viral DNA synthesis for accumulation to detectable levels. (iii) In immunofluorescence studies, the polyclonal UL 16/GST-specific antiserum was shown to stain the nucleus of infected cells at 18 hr after infection in areas containing high concentrations of HSV capsid proteins. These nuclear compartments have been described previously as viral assemblons (Ward et al., J. Virol. 70, 4623-4631, 1996) and are distinct from compartments containing replicating DNA. Localization within assemblons argues for a role of UL 16 encoded protein in capsid assembly or maturation. (iv) At 22 hr after infection, UL 16-specific immunofluorescence was present in both the nucleus and the cytoplasm. (v) Consistent with the change in localization at late times after infection, the UL 16 protein was found to be a component of purified virions.