We applied the technique of DNA amplification with the polymerase chain reaction to nine aqueous humor and five vitreous samples from HIV-1-infected patients with clinically diagnosed cytomegalovirus retinitis. For the amplification, recently published primers specific for herpes simplex virus (HSV), varicella zoster virus (VZV) and cytomegalovirus (CMV-1) were used. Additionally, a newly developed primer pair specific for the main immediately early gene of CMV (CMV-2) was selected and compared with the published one. All primers were tested on noninfected and HSV-, VZV- and CMV-infected human fibroblast cell culture supernatant, thereby excluding cross-reactivity of the chosen primers. In none of 13 aqueous humor and six vitreous samples of healthy controls was any viral DNA amplified. Using the CMV-1 primers, we detected CMV DNA in five of nine aqueous humor and three of five vitreous samples amplifying a DNA fragment 435 base pairs in length. With the CMV-2 primers, we detected a CMV DNA fragment with a length of 110 base pairs in eight of nine aqueous humor and in four of five vitreous samples. Additionally, CMV DNA was found in three of nine urine and two of nine saliva specimens. Both CMV and HSV DNA were amplified in one aqueous sample. Varicella DNA was not detected in any of the specimens. Thus, the polymerase chain reaction is more sensitive than other comparable diagnostic tests and may provide an alternative to conventional virus isolation and in situ hybridization techniques for the laboratory diagnosis of viral ocular disease.