A method based on manipulation of the human cytomegalovirus genome in a bacterial artificial chromosome was developed to determine the role played by 6 UL97 mutations of unknown significance. These mutations were found in blood samples from solid-organ transplant recipients in a trial comparing valganciclovir and oral ganciclovir prophylaxis. Recombinant viruses containing UL97 mutations P405L, A427V, M550I, A582V, Y617H, and A674T were generated in a bacterial system. Viral stocks were subsequently reconstituted in human fibroblasts, and ganciclovir susceptibilities were tested using a plaque-reduction assay. All recombinant viruses containing these unknown mutations were found to be susceptible to ganciclovir.