Drug resistance limits the efficacy of long-term antiviral therapy for cytomegalovirus (CMV) disease in severely immunosuppressed hosts. Although the advent of combination antiretroviral therapy has reduced the frequency of invasive CMV disease, it continues as a problem in those failing anti-HIV therapy and in transplant recipients who develop primary CMV infection. In the past few years we have developed a reasonable understanding of the common mutations in the CMV UL97 phosphotransferase and UL54 DNA polymerase genes that confer resistance to the current systemic anti-CMV drugs ganciclovir, foscarnet and cidofovir. This information is already widely used for genotypic diagnosis of CMV resistance. Some gaps in our knowledge remain. There are probably additional resistance mutations in lesser-known regions of UL97 and UL54, or in other viral genes involved in DNA replication. Yet other mutations seen in isolates from treated subjects probably confer little or no resistance. Specific resistance mutations seem to confer varying degrees of resistance to the same drugs when assessed in different viral genetic backgrounds. The effect of mutations on CMV replication ("fitness") has not been systematically investigated. A new class of anti-CMV benzimidazole riboside drugs offers potential therapeutic advantages in potency, oral bioavailability and different mechanisms of antiviral action from previous drugs. Exploring CMV strains resistant to these drugs will be important not only for therapeutic monitoring but for future drug development as well. Accordingly, the specific aims for the upcoming period are (1) to study additional viral mutations that appear to confer resistance and cross-resistance to current anti-CMV drugs, by transfer of the mutations to reference CMV strains, (2) investigate the phenotypic effects of single and multiple mutations on different viral genetic backgrounds, and (3) determine the genetic basis of CMV resistance to GW1263W94, a benzimidazole riboside drug now undergoing development for clinical use.