Purpose:Fundus autofluorescence was quantified (qAF) in subjects with healthy retinae using a standardized approach. The objective was to establish normative data and identify factors that influence the accumulation of RPE lipofuscin and/or modulate the observed AF signal in fundus images. Methods:AF images were acquired from 277 healthy subjects (age range: 5-60 years) by employing a Spectralis confocal scanning laser ophthalmoscope (cSLO; 488-nm excitation; 30°) equipped with an internal fluorescent reference. For each image, mean gray level was calculated as the average of 8 preset regions, and was calibrated to the reference, zero-laser light, the magnification, and the optical media density from normative data on lens transmission spectra. Relationships between qAF and age, gender, race/ethnicity, eye color, refraction/axial length and smoking status were evaluated as was measurement repeatability and the qAF spatial distribution. Results:qAF levels exhibited a significant increase with age. qAF increased with increasing eccentricity up to 10-15* from the fovea and was highest superotemporally. qAF values were significantly greater in females, and, compared to Hispanics, qAF was significantly higher in whites and lower in blacks and Asians. No associations with axial length and smoking were observed. For 2 operators, session-session repeatability was ±7.8% and ±9.7%. Agreement between the operators was ±10.1%. Conclusions:Normative qAF data are a reference tool essential to the interpretation of qAF measurements in ocular disease.