A real-time quantitative PCR-hybridisation assay was developed for the detection of human cytomegalovirus DNA in clinical material. The assay is based on a LightCycler (LC) and provides both rapid results (<1 h) and quantification over a broad dynamic range (2 x 10(3)-5 x 10(8) CMV DNA copies/ml). Given that the assay showed a 3-fold increase in sensitivity compared to detection of early antigen fluorescent foci (DEAFF) testing of urine samples, we investigated the practicality of testing surveillance such specimens from immunocompromised patients at risk of CMV disease. Over a 12-month period, CMV DNA was detected in 81 (7%) of 1154 urine samples examined. A total of 28 patients tested positive; urine viral loads were higher in 13 infants being investigated for suspected congenital infection (median 1.6 x 10(5) copies/ml) compared with 15 transplant recipients (median 9 x 10(3) copies/ml). Urine samples could be tested directly without processing such that results were available in <1h. Real-time PCR provided information on the quantification of CMV DNA in urine and proved a reliable method for the surveillance of immunocompromised patients at risk of CMV disease. This approach should facilitate a better understanding of the epidemiology and natural history of CMV disease. Moreover, LC-based quantitative PCR is a potentially valuable tool for the management of CMV disease; assisting with the prompt initiation of treatment and assessing therapeutic response.