Orf virus, a member of the Parapoxvirus genus, causes a contagious pustular dermatitis in sheep, goats, and humans. Previous studies have demonstrated the activity of (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC; cidofovir; Vistide) against orf virus in cell culture and humans. We have evaluated a broad range of acyclic nucleoside phosphonates (ANPs) against several orf virus strains in primary lamb keratinocytes (PLKs) and human embryonic lung (HEL) monolayers. HPMPC, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6- diaminopurine (HPMPDAP), and (R)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine (HPMPO-DAPy) were three of the most active compounds that were subsequently tested in a virus yield assay with PLK and HEL cells by virus titration and DNA quantification. HPMPC, HPMPDAP, and HPMPO-DAPy were evaluated for their activities against orf virus replication in organotypic epithelial raft cultures from differentiated PLK cells. At the highest concentrations (50 and 20 microg/ml), full protection was provided by the three drugs, while at 5 microg/ml, only HPMPDAP and HPMPC offered partial protection. The activities of the three compounds in the raft culture system were confirmed by quantification of infectious virus and viral DNA. These findings provide a rationale for the use of HPMPC and other ANPs in the treatment of orf (contagious ecthyma) in humans and animals.