Objective: To investigate the origin of signals from scanning laser Doppler flowmetry (SLDF) and the influence of axial scan depth on the measurement of blood flow in the rat retina.Methods: We performed SLDF in 5 adult Sprague-Dawley rats using a specially modified Heidelberg retina flowmeter. Axial scans were obtained from +2 diopters (D) to -3 D (in steps of 0.25 D) or from +1 D to -2 D ( in steps of 0.125 D) relative to the retinal surface. Fluorescein isothiocyanate-dextran angiograms were obtained in whole-mounted retinas to visualize the angio-architecture and identify measurement locations in the SLDF flow maps. Axial SLDF flow profiles were obtained in an artery, vein, arteriole, venule, and capillary bed using the mean blood flow values in 2 x 2 -, 4 x 4 -, and 10 x 10 - pixel measurement windows.Results: The SLDF images showed good correspondence with the angiograms and resolution to third-order arterioles and venules; however, neither the superficial nor deep capillary circulations were visualized. Flow was imaged from large choroidal vessels. Measured flow from capillaries was independent of depth and indistinguishable from background levels.Conclusion: The technique of SLDF images blood flow in larger retinal vessels but not in capillaries. Clinical Relevance: Scanning laser Doppler flowmetry may not reliably measure capillary blood flow.