Cytomegalovirus (CMV) isolates from subjects who received ganciclovir therapy were tested for susceptibility to ganciclovir by a plaque reduction assay. Results were correlated with restriction enzyme and sequence analysis of the CMV UL97 phosphotransferase gene. Of the 30 isolates, 20 had one or more mutations in UL97 affecting amino acid encoding at codons 460, 520, or 591-596. All 20 were resistant to ganciclovir, with an IC50 of > 6.0 microM (range, 6.7-50.0). The remaining 10 isolates had no mutations at these loci; 8 were susceptible to ganciclovir while the other 2 were borderline resistant (IC50s, 6.6 and 7.2 microM). None of 40 control CMV isolates from untreated subjects contained any amino acid changes at these loci. The three most common mutations at codons 460, 594, and 595 were detected by restriction digest analysis in 16 (80%) of 20 isolates and in 16 (73%) of 22 isolates with ganciclovir IC50s > 6.0 microM. These results indicate that the majority of ganciclovir-resistant clinical isolates contain diagnostically useful mutations in UL97.