We performed polymerase chain reaction (PCR) for detection of cytomegalovirus (CMV), varicella-zoster virus (VZV), herpes simplex virus (HSV), and Toxoplasma gondii DNA in aqueous humor from 15 patients who were infected with human immunodeficiency virus (HIV) and who had retinitis of unclear origin; these patients were selected from among 820 patients evaluated by ophthalmoscopic examination. On the basis of the final response to treatment, CMV, VZV, and T. gondii retinitis was diagnosed in 5, 2, and 4 of the 15 patients, respectively. No final etiologic diagnosis was reached for four patients. All 5 patients with CMV retinitis were CMV DNA-positive. 1 of 2 patients with VZV retinopathy were VZV DNA-positive, and 3 of 4 patients with T. gondii retinitis were T. gondii DNA-positive. All PCR assays of aqueous humor from the four patients without infectious retinitis were negative. PCR assay of aqueous humor is helpful in the etiologic diagnosis of retinitis of unclear origin in HIV-infected patients.