The herpes simplex virus (HSV-1) UL15 gene encodes one of the six viral gene products required for viral DNA cleavage and packaging. UL15 is a spliced gene and encodes two separately translated proteins, UL15 and UL15.5. Sequence analysis reveals that UL15 shares homology with gp 17, the large catalytic subunit of the bacteriophage T4 terminase, a protein which cleaves the polymeric T4 DNA into monomers. Both proteins contain a putative ATP binding motif known as the Walker A and B boxes. In this report, immunofluorescence was used to show that UL15 localizes to the nucleus in the absence of any other viral proteins; this indicates that UL15 contains its own nuclear localization signal. In addition, we found that UL15 colocalizes with replication compartments at early times (6 h postinfection). Since, at this time, preformed capsids as well as other cleavage and packaging proteins are also recruited to replication compartments, it seems likely that cleavage and packaging occurs in the same compartments in which DNA synthesis occurs. Also in this report, we have investigated UL15.5, the N-terminally truncated gene product of the UL15 open reading frame (ORF). The start codon has been mapped to Met443 within the UL15 ORF. Furthermore, we have shown that plasmids containing a UL15.5 knockout mutation still complement the growth of UL15 insertion mutant viruses, indicating that UL15.5 is not required for viral growth in cell culture. Last, we constructed a UL15 mutant, UL15C(G263A), in which the invariant Gly263 in the Walker box A of the ATP binding motif (GKT) was substituted with an alanine. We show that the mutant gene fails to support the growth of UL15 insertion mutant viruses, indicating that the putative ATP binding motif of UL15 is indispensable for its function.