Objectives This was a phase II trial to assess flurpiridaz F 18 for safety and compare its diagnostic performance for positron emission tomography (PET) myocardial perfusion imaging (MPI) with Tc-99m single-photon emission computed tomography (SPECT) MPI with regard to image quality, interpretative certainty, defect magnitude, and detection of coronary artery disease (CAD) (≥50% stenosis) on invasive coronary angiography (ICA). Background In pre-clinical and phase I studies, flurpiridaz F 18 has shown characteristics of an essentially ideal MPI tracer. Methods One hundred forty-three patients from 21 centers underwent rest-stress PET and Tc-99m SPECT MPI. Eighty-six patients underwent ICA, and 39 had low-likelihood of CAD. Images were scored by 3 independent, blinded readers. Results A higher percentage of images were rated as excellent/good on PET versus SPECT on stress (99.2% vs. 88.5%, p < 0.01) and rest (96.9% vs. 66.4, p < 0.01) images. Diagnostic certainty of interpretation (percentage of cases with definitely abnormal/normal interpretation) was higher for PET versus SPECT (90.8% vs. 70.9%, p < 0.01). In 86 patients who underwent ICA, sensitivity of PET was higher than SPECT (78.8% vs. 61.5%, respectively, p = 0.02). Specificity was not significantly different (PET: 76.5% vs. SPECT: 73.5%). Receiver-operating characteristic curve area was 0.82 ± 0.05 for PET and 0.70 ± 0.06 for SPECT (p = 0.04). Normalcy rate was 89.7% with PET and 97.4% with SPECT (p = NS). In patients with CAD on ICA, the magnitude of reversible defects was greater with PET than SPECT (p = 0.008). Extensive safety assessment revealed that flurpiridaz F 18 was safe in this cohort. Conclusions In this phase 2 trial, PET MPI with flurpiridaz F 18 was safe and superior to SPECT MPI for image quality, interpretative certainty, and overall CAD diagnosis.