The diagnosis of cytomegalovirus retinitis (CMV-R) is difficult and usually based on clinical criteria or invasive diagnostic procedures. The purpose of this study was to investigate a possible association between CMV-R and specific anti-CMV antibodies in tears. Paired tear and serum samples were obtained from 96 individuals, which included 20 children with congenital CMV infection and chorioretinitis, 56 adults with retinitis with clinical signs suggestive of viral infection, and 20 healthy control subjects, and were tested for CMV antibodies using ELISA. The prevalence of anti-CMV antibodies in tears was found to be 80% (16/20) in children, 35% (20/56) in adults, and 5% (1/20) in control subjects. Furthermore, high antibody levels were detected in 35% (7/20) of children and 10.7% (6/56) of adults with retinitis, and were not found in control subjects. There was a strong association between high tear levels of anti-CMV antibodies and active ocular infection. No correlations were found between tear and serum antibodies. ELISA sensitivity was 80% and specificity 95%. Further studies are needed to compare the tear and intraocular levels of CMV-specific antibodies in patients with retinitis to find out if CMV antibody testing in tear fluid could substitute for more invasive diagnostic procedures.