We validate a modification of the sedimentation method for measuring fluorescent microspheres (FM) that improves the determination of regional cerebral blood flow (rCBF). Our FM method for rCBF determination is compared to the radioactive microspheres (RM) method for rCBF measurement by simultaneous injection of one radioactive and two fluorescent labeled doses, at two separate time points, into the left ventricle of a pig. The pig was killed, the brain and spinal cord removed, and divided into 92 pieces averaging 0.83 g. Our modifications to FM analysis by sedimentation includes. 2 instead of 1 week of autolysis, pellet washing with 1% Triton X-100 instead of 0.25% Tween 80, phosphate buffer addition during rinse, fluorescent dye extraction using 2-ethoxyethylacetate instead of 2-(2-ethoxyethoxy)ethyl acetate and polypropylene instead of glass tubes. Comparing rCBF using Sc46 RM, to yellow-green and orange FM, yielded mean differences of 0.026 and 0.021 ml/min per piece, respectively. Sn-113 RM compared to blue-green and scarlet FM gave mean differences of - 0.010 and 0.137 ml/min per piece, respectively. All RM-FM differences, except those for scarlet FM, are within acceptable limits. This assay provides a reliable method for determining rCBF. (C) 1999 Elsevier Science B.V. All rights reserved.