SPECT can provide valuable diagnostic and treatment response information in large-scale multicenter clinical trials. However, SPECT has been limited in providing consistent quantitative functional parametric values across the centers, largely because of a lack of standardized procedures to correct for attenuation and scatter. Recently, a novel software package has been developed to reconstruct quantitative SPECT images and assess cerebral blood flow (CBF) at rest and after acetazolamide challenge from a single SPECT session. This study was aimed at validating this technique at different institutions with a variety of SPECT devices and imaging protocols. Methods: Twelve participating institutions obtained a series of SPECT scans on physical phantoms and clinical patients. The phantom experiments included the assessment of septal penetration for each collimator used and of the accuracy of the reconstructed images. Clinical studies were divided into 3 protocols, including intrainstitutional reproducibility, a comparison with PET, and rest-rest study consistency. The results from 46 successful studies were analyzed. Results: Activity concentration estimation (Bq/mL) in the reconstructed SPECT images of a uniform cylindric phantom showed an interinstitution variation of 65.1%, with a systematic underestimation of concentration by 12.5%. CBF values were reproducible both at rest and after acetazolamide on the basis of repeated studies in the same patient (mean 6 SD difference, 20.4 6 5.2 mL/min/100 g, n 5 44). CBF values were also consistent with those determined using PET (26.1 6 5.1 mL/min/100 g, n 5 6). Conclusion: This study demonstrates that SPECT can quantitatively provide physiologic functional images of rest and acetazolamide challenge CBF, using a quantitative reconstruction software package.Key Words: 123 I-iodoamphetamine; cerebral blood flow; acetazolamide; SPECT; vascular reactivity; quantitation J Nucl Med 2010; 51: 1624 -1631 DOI: 10.2967 Current clinical practice using SPECT relies largely on interpretation of qualitative images reflecting physiologic function. Quantitative functional parametric images may be obtained by applying mathematic modeling to SPECT data corrected for attenuation and scatter. Quantitative regional cerebral blood flow (CBF) (1-3) and cerebral vascular reactivity (CVR) in response to acetazolamide challenge (4-6) have been obtained with these techniques. One major application of such quantitative SPECT (QSPECT) approaches is the evaluation of ischemic status in patients with occlusion or stenosis in their middle cerebral arteries, to provide prognostic information of the outcome of revascularization therapies (7). Quantitative analysis in SPECT has also been demonstrated in the assessment of binding potential for several neuroreceptor ligands (8,9), for the quantitative assessment of regional myocardial perfusion (10,11), and for the assessment of radioaerosol deposition and clearance in healthy and diseased lungs (12). However, providing the standardized quantitative approach...