A rapid and simple quantitative dot immunoassay for a cell-adapted reference strain of cytomegalovirus (CMV) and for wild strains of CMV present in clinical urine samples was developed. The assay was performed on nitrocellulose paper dotted with several dilutions of viral pellets free of cellular debris. Viral dilutions were treated with a monoclonal antibody to the major component of the viral capsid. To amplify the reaction, a three-dimensional complex of streptavidin and biotinylated horseradish peroxidase was used as the detector system. The dot immunoassay, which does not require cell cultures, yielded results within one day. A significant correlation was found between CMV titers obtained by dot immunoassay and CMV infectious units determined by immunoalkaline phosphatase staining of CMV-late antigen positive cells.