Based on sequence analysis, the protein encoded by the UL3 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) was predicted to contain an N glycosylation site and to be a glycoprotein. To determine if this prediction was correct, we cloned and expressed the DNA encoding the complete sequence of the UL3 ORF in a baculovirus expression system. Western blotting was done using polyclonal antibody raised against synthetic UL3 peptides. Two major baculovirus-UL3 expressed protein bands with apparent molecular weights of 30 kDa and 31 kDa, and two minor protein bands with apparent molecular weights of 29 kDa and 33 kDa were detected. None of the expressed UL3 protein species were susceptible to tunicamycin treatment, suggesting that they were not N-linked glycosylated. Cell fractionation studies indicated that the UL3 protein was localized in the cytoplasmic and nuclear portion of the cells, rather than the cell membrane, again suggesting a lack of glycosylation. In contrast, the baculovirus expressed UL3 protein was phosphorylated as judged by 32Pi-labeling. Immunoprecipitation followed by SDS-PAGE demonstrated a single 32Pi-labeled UL3 related band with an apparent molecular weight of 33 kDa, indicating that the UL3 protein was a phosphoprotein. Antibodies produced in mice vaccinated with baculovirus-UL3 protein reacted with two UL3 related HSV-1 bands on Western blots. These protein bands had apparent molecular weights of 27 and 33 kDa and presumably represent the unphosphorylated and phosphorylated forms of UL3.