BACKGROUND: Cytomegalovirus (CMV) infection in immunocompromised patients can lead to viremia associated with morbidity and mortality. Monitoring of viral loads in blood is critical for initiating and monitoring antiviral treatment. OBJECTIVES: Validate quantitative real-time PCR assay targeting the US17 and UL54 regions of the CMV genome for automated DNA and extraction and amplification. STUDY DESIGN: 3422 blood specimens from organ transplant recipients, including longitudinal specimens from 12 organ transplant recipients, were tested by CMV PCR and pp65 antigenemia. RESULTS: CMV PCR for both US17 and UL54, was more sensitive and detected CMV DNA earlier and for longer than the CMV pp65 antigenemia test. Using antigenemia results as a reference standard, an optimal cutoff of 500 normalized copies was calculated for both US17 and UL54 PCR targets based on high sensitivity, specificity, and positive and negative predictive values. CMV DNA levels tracked well with clinical symptoms, response to treatment, and antigenemia. CONCLUSIONS: Detection of persistent increases in CMV DNA levels above 500 normalized copies by this real-time PCR assay is indicative of symptomatic CMV disease in organ transplant recipients. Quantitative real-time PCR for CMV DNA can be used in lieu of antigenemia for monitoring CMV infection and determining when to initiate preemptive treatment.