Thallium-201 has been used extensively as a myocardial perfusion agent and to assess myocardial viability. Unlike other 99mTc-labeled agents such as 99mTc-sestamibi and 99mTc-tetrofosmine, the regional concentration of 201Tl varies with time, and its kinetics make it a potential candidate for estimating absolute physiologic parameters with kinetic model analysis. This article outlines a strategy for quantitative assessment of regional myocardial blood flow in man using 201Tl and dynamic single photon emission computed tomography (SPECT). Quantitatively accurate SPECT images that are proportional to the true radioactivity distribution are prerequisites for model-based kinetic analysis. Our technique for quantitative SPECT includes ordered-subset maximum likelihood-expectation maximization (ML-EM) reconstruction with transmission data-based attenuation correction and transmission-dependent convolution subtraction scatter correction. A three-compartment model was found to reproduce the observed regional time-activity curves well, and dog experiments demonstrated that influx rate constant (K1) values estimated from the dynamic SPECT data correlated well with absolute myocardial blood flow determined by in vitro microspheres for a physiologically wide range of flows. Several possible strategies for simplifying the study procedures, without compromising accuracy, are also presented, which should make absolute quantitation of regional myocardial blood flow feasible using 201Tl and a conventional SPECT camera in a clinical setting.