Herpes simplex virus (HSV) types 1 and 2 thymidine kinases (TK) are responsible for phosphorylation of antiherpes acyclonucleosides such as acyclovir (ACV) and 9-(4-hydroxybutyl)guanine (HBG). Related compounds, the N2-phenyl-9-(hydroxyalkyl)guanines, are devoid of direct antiviral activity, but potently inhibit the viral TKs and block viral reactivation from latency in vivo. The similarity in structure between the acyclonucleosides and TK inhibitors raised the question of the relevance of phosphorylation of certain of the latter analogs in their mechanisms of action. Using recombinant TKs and HPLC analysis of reaction mixtures, we report that the lead TK inhibitor N2-phenyl-9-(4-hydroxybutyl)guanine (HBPG) and its pentyl homolog (HPnPG) are excellent substrates for the enzymes, approaching the efficiency with which the natural substrate thymidine is phosphorylated, and significantly better than ACV or HBG. Other 9-hydroxyalkyl congeners are substrates for the enzymes, but with much poorer efficiency. HBPG triphosphate was a poor inhibitor of HSV DNA polymerase, the target of acyclonucleoside triphosphates, suggesting that phosphorylation of HBPG is not important in its mechanism of blocking viral reactivation in vivo. The fact that HBPG is an efficient substrate is consistent, however, with its binding mode based both on molecular modeling studies and x-ray structure of the HBPG:TK complex