Radionuclide angiography with technetium-99m ethyl cysteinate dimer (ECD) allows non-invasive estimation of absolute cerebral blood flow (CBF), either by graphical Patlak-Gjedde analysis (PGA) or by spectral analysis (SA). Other methods estimate CBF by means of single-point arterial or venous sampling. The aim of this study was to evaluate radionuclide scanning and single-point venous sampling as potential clinical non- to minimally invasive methods for CBF determination in a large set of carefully screened healthy volunteers over the adult age range. Eighty-three carefully screened healthy volunteers (20-81 years, 43 males, 40 females) underwent planar radionuclide angiography with 925 MBq 99mTc-ECD. After correction for camera dead-time loss, hemispheric CBF was calculated from brain perfusion indices (BPI): BPI(G) for PGA and BPI(S) for SA. Of the volunteers, 49 also underwent venous sampling 6 min post injection, from which the lipophilic octanol extraction fraction and hemispheric brain fractionation index (BFI) were determined. All datasets were correlated and evaluated as a function of age and gender. Intrasubject variability for the BPI measurements was assessed in 11 volunteers by repeat study within 2 weeks of the first acquisition. Graphical and spectral analysis BPIs were strongly correlated (R=0.846, P<0.00001). This correlation coefficient increased to R=0.903 for the 74 cases in which graphical analysis was not hampered by temporal tracer retention in cervicobrachial venous valves. The BFI was weakly correlated to both BPI indices (BPI(G): R=0.34, P=0.02; BPI(S): R=0.31, P=0.04). The right hemisphere showed significant asymmetry for BPI(S) (AI=2.7%+/-4.3%, P<0.001), in correspondence with previous 99mTc-ECD data. BPI(G), BPI(S) and BFI were all inversely related to age, with an increased gradient after the age of 55 years, while there was no significant gender difference. The ratio of BPI(G) to BIP(S), which is a measure of the cerebral extraction fraction for 99mTc-ECD, was not dependent on age. Intersubject variability was 15.5% for both radionuclide scanning-based methods and 18.2% for venous sampling, and in all cases was independent of age. A much lower intrasubject variability was observed for BPI(S) (7.2%) than for BPI(G) (12.6%). This study provides reference values for normal perfusion indices assessed by graphical and spectral analysis. The results also indicate that spectral analysis allows the most reproducible estimate of hemispheric perfusion by means of an operator-independent and objective approach. Whereas accurate calibration of normal BPI(S) values to hemispheric CBF with established methods needs to be performed, non-invasive calculation of regional absolute CBF using 99mTc-ECD is possible by application of a linearisation algorithm.