The quantitative determination of regional cerebral blood flow (rCBF) is important in certain clinical and research applications. The disadvantage of most quantitative methods using H(2)(15)O positron emission tomography (PET) is the need for arterial blood sampling. In this study a new non-invasive method for rCBF quantification was evaluated. The method is based on the washout rate of H(2)(15)O following intravenous injection. All results were obtained with Alpert's method, which yields maps of the washin parameter K(1) (rCBF(K1)) and the washout parameter k(2) (rCBF(k2)). Maps of rCBF(K1) were computed with measured arterial input curves. Maps of rCBF(k2*) were calculated with a standard input curve which was the mean of eight individual input curves. The mean of grey matter rCBF(k2*) (CBF(k2*)) was then compared with the mean of rCBF(K1) (CBF(K1)) in ten healthy volunteer smokers who underwent two PET sessions on day 1 and day 3. Each session consisted of three serial H(2)(15)O scans. Reproducibility was analysed using the rCBF difference scan 3-scan 2 in each session. The perfusion reserve (PR = rCBF(acetazolamide)-rCBF(baseline)) following acetazolamide challenge was calculated with rCBF(k2*) (PR(k2*)) and rCBF(K1) (PR(K1)) in ten patients with cerebrovascular disease. The difference CBF(k2*)-CBF(K1) was 5.90+/-8.12 ml/min/100 ml (mean+/-SD, n=55). The SD of the scan 3-scan 1 difference was 6.1% for rCBF(k2*) and rCBF(K1), demonstrating a high reproducibility. Perfusion reserve values determined with rCBF(K1) and rCBF(k2*) were in high agreement (difference PR(k2*)-PR(K1)=-6.5+/-10.4%, PR expressed in percentage increase from baseline). In conclusion, a new non-invasive method for the quantitative determination of rCBF is presented. The method is in good agreement with Alpert's original method and the reproducibility is high. It does not require arterial blood sampling, yields quantitative voxel-by-voxel maps of rCBF, and is computationally efficient and easy to implement.