In order to identify functional regions of the human cytomegalovirus protein pUL97 (i) different 5' fragments of the UL97 open reading frame (ORF) were fused to the coding region of the green fluorescent protein and (ii) recombinant vaccinia viruses (rVV) were generated carrying two full-length and 11 mutated UL97 ORFs. The results indicated the presence of an N-terminal region within pUL97 which changed the intracellular distribution of the fusion proteins. pUL97 was localized in the nucleus, but not in the nucleoli, and was detected in the nuclear matrix fraction. Expression of all pUL97 mutants could be confirmed by Western blot analysis. pUL97-associated ganciclovir (GCV) phosphorylation in rVV-infected cells, determined quantitatively by HPLC analysis, was abolished completely using individual UL97 deletion mutants. Phosphorylation of full-length and some of the mutated pUL97 was detected in cells infected with the rVVs. The UL97 constructs carrying point mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V, and the 4 aa deletion 590AACR593, also resulted in decreased but not abolished phosphorylation of GCV in the rVV system, whereas the phosphorylation of pUL97 itself was not influenced. The rVV system is a suitable method for quantitatively testing the functional relevance of pUL97 mutations.