OBJECTIVE To image and quantify the spatial distribution of fundus autofluorescence in normal subjects, to determine its age dependence, and to document the deviation from normal in patients with age-related macular disease. METHODS Using a confocal laser scanning ophthalmoscope (cLSO), the intensity and spatial distribution of fundus autofluorescence was studied in 33 normal subjects, 97 eyes with drusen only, and 111 eyes with visual loss caused by age-related macular disease. RESULTS Fundus autofluorescence intensity in normal subjects was highest at the posterior pole and dipped at the fovea. Autofluorescence increased with age at the posterior pole. Fundus in eyes with age-related maculopathy showed localized high autofluorescence that did not correspond with drusen. Linear pigmentation at the level of the retinal pigment epithelium (RPE), whether detached or flat, fluoresced brightly, whereas plaques of melanin did not. Areas of low and high levels of autofluorescence were seen in lesions containing choroidal new vessels. In areas of geographic atrophy, autofluorescence was low. CONCLUSIONS The spatial distribution of background fundus autofluorescence and the correlation of autofluorescence with age in normal subjects imply that autofluorescence is derived from lipofuscin at the level of the RPE. Focal accumulation of autofluorescent material occurs at the level of the RPE in patients with drusen, but the drusen do not show marked increases in autofluorescence. It is likely that melanolipofuscin accounts for the high levels of autofluorescence, corresponding to linear pigmentation at the level of the RPE. Low-intensity autofluorescence occurs in the presence of retinal photoreceptor loss, and variable levels over disciform lesions probably relate to variations in metabolic activity of the RPE.