In situ hybridization (ISH) for identification of infectious replicative cytomegalovirus (CMV) in cell culture microplates (96 microwells) infected by clinical specimens was tested by using a biotin-labeled DNA probe and an avidin-alkaline phosphatase conjugate. A total of 395 specimens were examined by using ISH and a monoclonal antibody (MAb) specific for an early antigen of CMV. Of 47 specimens that gave a positive signal for CMV by ISH, 33 were confirmed virus positive by MAb staining. Of 141 blood samples tested, 4.96% were positive by ISH, and 0.7% were positive by the MAb technique. ISH shows 40% more sensitivity than MAb staining. This technique should be widely applicable for the specific identification of viral isolates (e.g., herpesvirus, myxovirus, paramyxovirus, and enterovirus) in cell culture 96-microwell microplates, thereby making it feasible to screen a larger number of samples than is possible with classical methods using conventional culture tubes, shell vials, or 24-well plates.