Twenty-seven (10%) of 271 infants and children with acute otitis media (AOM) were found to be infected with cytomegalovirus (CMV) or herpes simplex virus type 1 (HSV). CMV or HSV, alone or in combination with bacteria or other viruses, was isolated from the middle ear fluid (MEF) of 10 patients. In three cases, CMV alone was isolated from the MEF, and in one case, HSV alone was isolated. One of the CMV cases involved an acute primary or reactivation of CMV infection, with CMV-bacterial otitis and conjunctivitis as major manifestations. One patient with AOM and stomatitis had purulent otitis associated with the presence of HSV in MEF, with no other bacterial or viral pathogens noted in MEF or nasal wash specimens. While most patients with CMV infection were probably asymptomatic excreters at the time of development of AOM, CMV did enter the middle ear. The presence of CMV in MEF was prolonged, and the patients continued to have clinical signs of otitis despite negative bacterial cultures. Among patients with bacterial otitis, a higher proportion of those who had CMV found only in nasal wash specimens had persistent bacteria in MEF, compared with those who were concurrently infected with other viruses (57% vs. 19%; P less than .04). This report is the first to suggest an etiologic role for CMV and HSV in AOM.