The ubiquitous nature of HHV-6 and its genomic relationship with cytomegalovirus led us to evaluate an etiological link between HHV-6 and AIDS-associated retinitis in a prospective study. HHV-6 infection was studied in patients with AIDS-associated retinitis and in two control populations. Eye pairs were obtained at necropsy from nine patients with AIDS-associated retinitis, four human immunodeficiency virus (HIV)-sero-positive patients with normal fundus examination and three HIV-seronegative patients. HHV-6 infection was detected by polymerase chain reaction (PCR), in situ hybridization and immunohistochemistry. Human cytomegalovirus (CMV) and HIV-1 infections were detected in parallel by the same methods. HHV-6 infection was detected in three cases of AIDS-associated retinitis. In two of these patients, HHV-6 infection was detected both by immunohistochemistry and PCR while in the third case it was detected by in situ hybridization and PCR. In the three patients, fundus examination showed bilateral retinitis in two of them and unilateral retinitis in one of them. HHV-6 infection was not detected in the retina of the two control groups. CMV was also detected in the three cases positive for HHV-6 by all three methods. HIV DNA was detected by PCR in two of three cases and was confirmed in one of these cases by in situ hybridization. These results confirm that HHV-6 infects the retina but suggests that HHV-6 does not have an exclusive causative role in AIDS-associated retinitis, since CMV coinfection of the retina was detected in all three of the patients positive for HHV-6.