OBJECTIVE Fundus autofluorescence (FAF) affects the overlying absorptive retinal pigments within the eye and can potentially be used to assess their density. This study reports a clinical application of FAF in measuring photopigments by scanning laser ophthalmoscopy (SLO). METHODS The study group comprised 20 healthy subjects, 4 patients with branch retinal artery occlusion (BRAO), 3 with macular hole, 3 with branch retinal vein occlusion (BRVO), and 4 with resolved central serous chorioretinopathy (CSC). Serial FAF images were taken during exposure to light. The intensity of the FAF was measured at the site of the macular hole or the photocoagulation laser burn in the eyes with BRVO. The autofluorescence optical density difference (fODD) was measured from the FAF images and mapped to elucidate the topographic pattern. RESULTS The autofluorescence intensity showed little change at the sites of the macular holes or photocoagulation burns during exposure to light. The fODD was smallest at the center of the fovea and gradually increased with the eccentricity within 270 x 270 pixels around the fovea in healthy subjects. The amplitude of the fODD did not change in the area affected with BRAO in comparison to the unaffected area. By contrast, the fODD decreased in the area of resolved serous retinal detachment in the eyes with CSC. CONCLUSIONS In eyes with retinal disease, measuring the autofluorescence intensity using SLO is a feasible method of assessing the changes in the photopigments. Further studies comparing this approach with conventional methods for examining photopigments are needed.