Virus-associated DNA polymerase activity has recently been proposed for the detection of human cytomegalovirus (HCMV) in urine, a method that should allow rapid and quantitative determination of the viral load. In this report, the virus-associated DNA polymerase activity recovered from the urine of a group of patients shedding HCMV was measured using a poly(dA) oligo(dT) 12-18 synthetic template after polyethylene glycol precipitation of the virions. Detection of virus-associated DNA polymerase activity was compared to the classical methods most widely used to diagnose HCMV shedding in urines such as virus culture followed by indirect immunofluorescence and pp65 gene-specific polymerase chain reaction. Although less sensitive than the polymerase chain reaction and cross-reactive with other herpesvirus DNA polymerases, the activity measured in the urine samples was correlated with the number of positive nuclei found in shell vials (r = 0.89). The diagnostic threshold of the assay could be placed between 50 and 100 fluorescent nuclei per shell with a diagnostic sensitivity of 56%. Being simple and quantitative, the measurement of virus-associated DNA polymerase activity could be of value in some clinical conditions where it is necessary to assess viral load in urine. This method is proposed as an alternative to more laborious quantitative assays and to support qualitative polymerase chain reaction.