We have raised rabbit polyclonal antisera against a His-tagged herpes simplex virus type 1 (HSV-1) UL16 fusion protein, one of which very specifically reacted with 40 kDa and 41 kDa proteins in the lysates of HSV-1 and HSV-2-infected cells, respectively. Since its reactivity to the 41 kDa protein was clearly eliminated by pre-adsorption with E. coli lysates expressing the UL16 fusion protein, the antiserum was used to characterize the UL16 products of HSV-2. The HSV-2 UL16 protein was produced at the late phase of infection in a manner highly dependent on viral DNA synthesis and was distributed in both the nuclei and the cytoplasma of infected cells. In immunofluorescence studies, the UL16-specific fluorescence in the nuclei was shown to be detected as small discrete granules. On the other hand, the cytoplasmic fluorescence was diffusely distributed around the nucleus at 8 h postinfection but, at later times of infection, it was mainly detected as a mass at a perinuclear region. The analysis on its association with capsids has revealed that the UL16 protein copurified with C capsids but not B and A capsids, and that the association with C capsids was not tight. Moreover, our experiments have shown that a detectable level of the UL16 protein was not associated with extracellular virions, and that the partially purified UL16 proteins had a DNA-binding activity. These observations are consistent with the hypothesis that the UL16 protein plays a role in capsid maturation including DNA packaging/cleavage. We have also determined the complete nucleotide sequence of the HSV-2 UL16 gene and found that a nonstandard initiation codon may be used for its translation.