[Truncated abstract] Purpose: To improve our ability to interpret and validate Heidelberg Retina Flowmeter (HRF) flow images by recording flow measurements from specific regions of the retinal vasculature in in-vitro and in-vivo eye models in which retinal blood flow can be manipulated. Methods: The first experiments utilised an isolated perfused pig eyes perfused with a 50%/50% Krebs/RBC solution at known flow rates ranging from zero to 300 μl/min. After HRF image acquisition, the retinal vasculature was perfused with fluorescein isothiocyanate (FITC), for fluorescence microscopy. Using the standard HRF software and a 10×10 pixel measurement window, flow rates were measured from a retinal artery, vein, arteriole, venule, and the retinal capillary bed and a capillary-free-zone. The relationship between HRF measured flow and perfusion flow in the different measurement regions was determined. For the second study, HRF flow images were acquired at retinal sites in Brown Norway rats over a range of focus levels before and after cessation of retinal blood flow by laser-induced central retinal artery occlusion. Using the 10×10 pixel and 4×4 pixel measurement windows, HRF measured flow was performed in retinal artery, vein, arteriole, capillary and choroidal vessel locations. The relationship between HRF measured flow and focus depth was determined for each location before and after central retinal artery occlusion. At the conclusion of each experiment the effect of reduction of systemic blood pressure (by exsanguination) on HRF flow measurements in choroidal vessels and in locations without visible choroidal vessels was assessed. Finally, HRF flow measurements were acquired after euthanasia, to determine the HRF flow signal which was still present under zero biological flow conditions (the zerooffset). Results: In the isolated perfused pig eye experiments it was found that the HRF flow response varied according to vascular location. At zero perfusate flow, HRF flow was consistently greater than zero at all locations, averaging 171.9 ± 44.7 AU (n=97), representing background noise. Arteries and veins yielded the highest HRF measured flow values, but the relationship between HRF measured flow and perfusate flow was not linear. In arterioles the HRF flow was more linear over a broader range of perfusate flow rates but the peak flow signal was an order of magnitude smaller than that in arteries and veins. Both the linearity and magnitude of the flow signal in venules was less than that in arterioles.