Mutations in both the viral phosphotransferase gene, UL97, and the DNA polymerase gene, UL54, have been shown to confer ganciclovir resistance to cytomegalovirus (CMV). Moreover, UL54 alterations have been associated with in vitro cross-resistance of CMV to cidofovir. To investigate the relative significance of UL97 versus UL54 alterations in conferring antiviral resistance, phenotypic and genotypic characterization of 28 ganciclovir-resistant clinical CMV isolates was undertaken. Isolates were either low-level ganciclovir-resistant, which have ganciclovir ID50 values > or =8 microM and <30 microM and sensitivity to cidofovir, or high-level ganciclovir-resistant, which have ganciclovir ID50 values > or =30 microM and cross-resistance to cidofovir. Low-level ganciclovir-resistant isolates were associated with UL97 alterations and short periods of ganciclovir treatment, while high-level ganciclovir-resistant isolates were associated with both UL97 and polymerase alterations and were cultured after extended ganciclovir therapy.