In this study, we continued to study antiherpetic properties of acyclovir 5′-hydrogenphosphonate (Hp-ACV) in cell cultures and animal models. Hp-ACV was shown to inhibit the development of herpetic infection in mice induced by the HSV-1/L2strain. The compound suppressed replication of both ACV-sensitive HSV-1/L2and ACV-resistant HSV-1/L2/R strains in Vero cell culture. Viral population resistant to Hp-ACV (HSV-1/L2/RHp-ACV) was developed much slower than ACV-resistant population. The analysis of Hp-ACV-resistant clones isolated from the HSV-1/L2/RHp-ACVpopulation demonstrated their partial cross-resistance to ACV. The mutations determining the resistance of HSV-1 clones to Hp-ACV were partly overlapped with mutations defining ACV resistance but did not always coincide. HSV-1/L2/RHp-ACVherpes virus thymidine kinase is shortened from the C-terminus by 100 amino acid residues in length. © 2009 John Wiley & Sons A/S.