Although there is currently no FDA approved antiviral treatment for adenovirus (Ad) infections, the broad spectrum antiviral cidofovir (CDV) has demonstrated potent inhibitory activity against many Ad serotypes in vitro and in an in vivo ocular replication model. The clinical potential of CDV prompted the assessment for the emergence of CDV resistance in Ad5. Serial passage of Ad5 in increasing concentrations of CDV resulted in derivation of four different Ad5 variants with increased resistance to CDV. CDV resistance was demonstrated by ability to replicate viral DNA in infected cells at CDV concentrations that inhibit the parental virus, by ability to form plaques in CDV concentrations of >20 microg/ml and by increased progeny release following infection and growth in media containing CDV. Using marker rescue, the loci for CDV resistance in variant R1 was shown to be mediated by one residue change L741S, one of two mutations within the R1 encoded DNA polymerase. The CDV-resistant variants R4, R5 and R6 also contained mutations in their respective DNA polymerase sequences, but these were different from R1; variant R4 contained two changes (F740I and V180I), whereas both R5 and R6 variants contained the non-conserved mutation A359E. R6 contained additional alterations L554F and V817L. The location of the R1 change is close to a region of the DNA polymerase which is conserved with other polymerases that is predicted to involve nucleotide binding.