Bidirectional laser Doppler velocimetry (BLDV) for the measurement of retinal blood flow was validated in six anaesthetised minipigs, by comparing BLDV derived results with those obtained using radioactively labelled microspheres (RLM). The mean velocity of blood (Vmean) was calculated from the maximum red blood cell velocity measured by BLDV. Volumetric flow rate was determined from Vmean and vessel diameter, measure from monochromatic fundus photographs. Total retinal blood flow (TRBF) was calculated by summating flow values obtained for each retinal vein draining into the optic disc. A significant correlation was found between the TRBF results obtained by the two techniques (r = 0.99, p less than 0.001). The BLDV results were between 3-35 microliters/min lower than the corresponding RLM results (p = 0.05). Values of 57 +/- 24 microliters/min and 76 +/- 34 microliters/min were obtained for TRBF using the BLDV and RLM techniques respectively. Reproducibility studies with BLDV were also performed in six anaesthetised pigs over three hours and in six normal human volunteers over two hours and two weeks. No significant difference between measurements was found with time. Ninety five percent confidence limits of +/- 9.8% for the six pigs and +/- 8.9% for the six human volunteers were found for measurements on the same day and at two weeks. We conclude that with a sample size of six, changes in flow of approximately 20% can be detected using BLDV and monochromatic fundus photography.