Four human cytomegalovirus (HCMV) isolates from four different AIDS patients treated with both ganciclovir and foscarnet and not responding clinically to antiviral treatment, were studied in order to verify the occurrence of double resistance to both drugs, and to define whether single or multiple HCMV strains could be responsible for the double resistance. Peripheral blood leukocytes (PBL), the relevant conventional viral isolates, and plaque-purified strains from all four patients were examined by antiviral drug susceptibility testing by an immediate-early antigen plaque reduction assay and by restriction fragment length polymorphism (RFLP) analysis using polymerase chain reaction (PCR)-amplified multiple genome regions and endonucleases. All four HCMV strains had a high level of resistance to both ganciclovir and foscarnet. A single HCMV strain was shown to be responsible for the dual resistance in each patient. HCMV strain identity and uniqueness were shown for each of the four patients in blood samples, viral isolates, and plaque-purified strains. In addition, in two patients the same single HCMV strain shifted progressively from drug sensitivity to ganciclovir and then to ganciclovir-foscarnet resistance. These findings document that resistance to both ganciclovir and foscarnet of HCMV strains recovered from blood of AIDS patients represents an emerging problem. Although it is known that multiple HCMV strains may cocirculate in the blood of AIDS patients, single strains appear to be responsible for the dual resistance. Molecular mechanisms responsible for the double resistance of the four reported strains are under study.