The aim of this study was to develop a new method for quantifying regional cerebral blood flow (rCBF) with technetium-99m ethyl cysteinate dimer (ECD) and single-photon emission tomography (SPET). Employing a three-compartment model, we introduced a parameter, regional brain fractionation index (BFI), that reflects rCBF values and is obtained by a single SPET scan at optimum time T (min) after tracer injection and the integral of arterial input. By analysing the dynamic SPET and arterial blood sampling data of 15 subjects, including the results of acetazolamide challenges, with the graphical plot method, optimum time T was determined to be approximately 20 min post injection. Regional BFI values of each subject were calculated from the single SPET data at 20 min and arterial input. The relationship between the values of regional BFI and rCBF obtained by xenon-133 inhalation SPET was analysed by approximation with an exponential function, resulting in good agreement (r = 0.907). In the present method, I CBF values were determined from regional BFI values by using the inverse exponential function as a non-linear regression curve. To validate the method, we applied it to six other subjects, in whom acetazolamide challenges were also performed. In comparing rCBF values thus obtained and those obtained by Xe-133 inhalation SPET, we found a good correlation (r = 0.901) with an inclination approximating 1 (= 1.02) and without underestimation of rCBF in the high-flow range. Since the present method does not require dynamic planar imaging or dynamic SPET scanning, it can be applied to any type of SPET scanner and is useful in clinical SPET studies.