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Background The accuracy of absolute myocardial blood flow (MBF) from dynamic contrast-enhanced cardiac computed tomography acquisitions has not been fully characterized. We evaluate computed tomography (CT) compared with rubidium-82 positron emission tomography (PET) MBF estimates in a high-risk population. Methods In a prospective trial, patients receiving clinically indicated rubidium-82 PET exams were recruited to receive a dynamic contrast-enhanced cardiac computed tomography exam. The CT protocol included a rest and stress dynamic portion each acquiring 12 to 18 cardiac-gated frames. The global MBF was estimated from the PET and CT exam. Results Thirty-four patients referred for cardiac rest-stress PET were recruited. Of the 68 dynamic contrast-enhanced cardiac computed tomography scans, 5 were excluded because of injection errors or mismatched hemodynamics. The CT-derived global MBF was highly correlated with the PET MBF (r=0.92; P<0.001) with a mean difference of 0.7±26.4%. The CT MBF estimates were within 20% of PET estimates ( P<0.02) with a mean of (1) MBF for resting flow of PET versus CT of 0.9±0.3 versus 1.0±0.2 mL/min per gram and (2) MBF for stress flow of 2.1±0.7 versus 2.0±0.8 mL/min per gram. Myocardial flow reserve was -14±28% underestimated with CT (PET versus CT myocardial flow reserve, 2.5±0.6 versus 2.2±0.6). The proposed rest+stress+computed tomography angiography protocol had a dose length product of 598±76 mGy×cm resulting in an approximate effective dose of 8.4±1.1 mSv. Conclusions In a high-risk clinical population, a clinically practical dynamic contrast-enhanced cardiac computed tomography provided unbiased MBF estimates within 20% of rubidium-82 PET. Although unbiased, the CT estimates contain substantial variance with an standard error of the estimate of 0.44 mL/min per gram. Myocardial flow reserve estimation was not as accurate as individual MBF estimates.