Emergence of drug-resistant human cytomegalovirus (HCMV) strains is a substantial problem during treatment of HCMV infections in immunocompromised patients. The Z-isomers of 2-hydroxymethylcyclopropylidenemethyl adenine (synadenol) and guanine (synguanol) were previously shown to be potent inhibitors of AD169 and Towne HCMV reference strains and postulated to share a common phosphorylation pathway with ganciclovir (GCV) possibly involving the UL97-encoded phosphotransferase. Analysis of synadenol and synguanol susceptibility of a series of HCMV isolates from immunocompromised untreated patients and from patients with treatment failure due to the emergence of GCV- and foscarnet (PFA)-resistant HCMV strains demonstrated that synadenol and synguanol are potent inhibitors of clinical HCMV isolates and are highly effective against both GCV- and PFA-resistant isolates. These results together with those showing resistance of a UL97 knock-out HCMV mutant to GCV as well as synadenol and synguanol suggest the involvement of UL97 phosphotransferase in synadenol and synguanol anabolism but with a substrate specificity different from that of GCV.