Adenoviruses have been recognized as important pathogens in immunocompromised hosts. Particularly in pediatric allogeneic stem cell transplant recipients, the morbidity of the patients and mortality in those patients with disseminated infections have been found to increase over the last few years. Severe infections are predominantly but not exclusively caused by subgroup C adenoviruses. A multiplex real-time PCR assay using molecular beacons as probes was developed to enable monitoring of adenovirus DNA in those patients with simultaneous identification of subgroups. An internal control was coamplified in the multiplex PCR to check for the DNA isolation procedure as well as the presence of inhibitors in the clinical samples. The assay has been applied retrospectively in patient groups with different clinical outcomes of infection. In fatal cases, significantly higher adenovirus loads developed, exceeding even 10(11) copies/ml of serum or plasma. Patients with viral loads over 10(6) copies/ml appear to have an increased risk for fatal complications. This quantitative real-time PCR assay has been prospectively used clinically since 2002 to study the course of adenovirus infection. In addition, the assay provides objective start and end points of therapeutic interventions, including the clinically important evaluation of antiviral drugs.