The temporal expression of the UL97 gene product during human cytomegalovirus (HCMV) infection of human foreskin fibroblasts (HFF) and subcellular localization of this protein were analyzed by using a polyclonal antiserum raised against a truncated UL97 protein of 47 kDa. The UL97 protein was detectable 16 h after infection by Western blot (immunoblot) analysis. Since only reduced UL97 expression occurred in the presence of two inhibitors of DNA replication, phosphonoacetic acid and ganciclovir, we conclude that UL97 is an early-late gene, requiring DNA replication for maximum expression. By indirect immunofluorescence, the protein could be visualized in the nuclei of virus-infected HFF 22 h after infection. Nuclear localization of the UL97 protein was also detected in thymidine kinase-deficient 143B cells infected with a recombinant vaccinia virus containing the entire UL97 open reading frame (ORF), as well as in HFF transiently expressing the entire UL97 ORF under the control of HCMV major immediate-early promoter. However, transiently expressed 5'-terminal deletion mutants of the UL97 ORF in addition showed a cytoplasmic localization of the UL97 protein, confirming the presence of a nuclear localization site in the N-terminal region of the protein. Our high-pressure liquid chromatography analyses confirmed the ganciclovir phosphorylation by the UL97 protein, but no specific phosphorylation of natural nucleosides was observed, indicating that the UL97 protein is not a nucleoside kinase. During plaque purification of recombinant UL97-deficient HCMV, this virus was growth defective; hence, we presume that UL97 may be essential for the viral life cycle.