Background: We previously proposed a technique for quantitative measurement of rest and stress absolute myocardial blood flow (MBF) using a 2-injection single-scan imaging session. Recently, we validated the method in a pig model for the long-lived radiotracer 18 F-Flurpiridaz with adenosine as a pharmacological stressor. The aim of the present work is to validate our technique for 13 NH 3 . Methods: Nine studies were performed in 6 pigs; 5 studies were done in the native state and 4 after infarction of the left anterior descending artery. Each study consisted of 3 dynamic scans: a 2-injection rest-rest single-scan acquisition (scan A), a 2-injection rest/stress single-scan acquisition (scan B), and a conventional 1-injection stress acquisition (scan C). Variable doses of adenosine combined with dobutamine were administered to induce a wide range of MBF. The 2-injection single-scan measurements were fitted with our nonstationary kinetic model (MGH2). In 4 studies, 13 NH 3 injections were paired with microsphere injections. MBF estimates obtained with our method were compared with those obtained with the standard method and with microspheres. We used a model-based method to generate separate rest and stress perfusion images. Results: In the absence of stress (scan A), the MBF values estimated by MGH2 were nearly the same for the 2-radiotracer injections (mean difference: 0.067±0.070 mL·min −1 ·cc −1 , limits of agreement: [−0.070 to 0.204] mL·min −1 ·cc −1 ), showing good repeatability. Bland-Altman analyses demonstrated very good agreement with the conventional method for both rest (mean difference: −0.034±0.035 mL·min −1 ·cc −1 , limits of agreement: [−0.103 to 0.035] mL·min −1 ·cc −1 ) and stress (mean difference: 0.057±0.361 mL·min −1 ·cc −1 , limits of agreement: [−0.651 to 0.765] mL·min −1 ·cc −1 ) MBF measurements. Positron emission tomography and microsphere MBF measurements correlated closely. Very good quality perfusion images were obtained. Conclusions: This study provides in vivo validation of our single-scan rest-stress method for 13 NH 3 measurements. The 13 NH 3 rest/stress myocardial perfusion imaging procedure can be compressed into a single positron emission tomography scan session lasting less than 15 minutes.