Polyomavirus infections in humans are due to BK virus (BKV) and JC virus (JCV). Diseases associated with human polyomaviruses occur mostly in immunocompromised adults, e.g., progressive multifocal leukoencephalopathy (PML), caused by JCV, in AIDS patients and hemorrhagic cystitis and uretral stenosis, caused by BKV, in transplant recipients. No therapy is available for these diseases, which necessitates the development of chemical entities that are active against polyomaviruses. Several antiviral compounds were evaluated to determine their effects on the in vitro replication of mouse polyomavirus and the primate viruses simian virus 40 (SV40), SV40 PML-1, and SV40 PML-2. The activity of the different compounds was assessed by a cytopathic effect reduction assay and confirmed in a virus yield assay. Cidofovir [HPMPC; (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] and its cyclic counterpart emerged as the most selective antipolyomavirus agents. The 50% inhibitory concentrations for HPMPC were in the range of 4 to 7 micrograms/ml, and its selectivity index varied from 11 to 20 for mouse polyomavirus and from 23 to 33 for SV40 strains in confluent cell monolayers. Cell cytotoxicity was up to 15-fold greater in growing cells. Other acyclic nucleoside phosphonates (i.e., HPMPA; [(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine] and PMEG [9-(2-phosphonylmethoxyethyl)-guanine]) also showed some activity but had low selectivity. None of the other drugs tested against these animal viruses (i.e., acyclovir, ganciclovir, brivudine, ribavirin, foscarnet, and cytarabine) showed significant activity. Thus, HPMPC deserves further evaluation as a candidate drug for polyomavirus infections in the immunocompromised host.