PURPOSE: To determine whether cytomegalovirus (CMV) retinitis that responded poorly to intravenous ganciclovir therapy but responded to the ganciclovir implant was caused by virus with resistance mutations in the viral UL97 and UL54 genes. DESIGN: Retrospective chart review and laboratory-based experimental study. METHODS: Regions of the CMV UL97 and UL54 were amplified from the vitreous and analyzed for resistant mutations by a combination of DNA sequencing and restriction digestion. Vitreous from patients with AIDS and retinal infections other than CMV retinitis served as negative controls. RESULTS: We amplified all target regions of UL97 DNA and most target regions of UL54 DNA from eight eyes with CMV retinitis. In one eye we found a ganciclovir resistance mutation at base 1781 of the UL97 gene, predicting an alanine to valine mutation at codon 594. In a second eye we found a ganciclovir resistance mutation at base 2960 of the UL54 gene, predicting an alanine to glycine mutation at codon 987. In two additional eyes, both from patients with bilateral retinitis, we found UL54 mutations that are likely to confer resistance to ganciclovir but have not been previously described. In both of these patients the UL54 genotype differed between the two diseased eyes. CONCLUSIONS: Failure to control CMV retinitis with intravenous ganciclovir does not necessarily imply infection with a virus having a known mutation that confers drug resistance. The ganciclovir implant can be an effective therapy for CMV retinitis caused by virus with certain UL97 and UL54 resistance mutations. Cytomegalovirus UL54 genotypes can differ between eyes in patients with bilateral retinitis.