INTRODUCTION: X-ray based digital subtraction angiography (DSA) is a common clinical imaging method for vascular morphology and function. Coronary artery characterization is one of its most important applications. We show that bi-plane DSA of rat coronary arteries can provide a powerful imaging tool for translational safety assessment in drug discovery. METHODS: A novel, dual tube/detector system, constructed explicitly for preclinical imaging, supports image acquisition at 10 frames/s with 88-micron spatial resolution. Ventilation, x-ray exposure, and contrast injection are all precisely synchronized using a biological sequence controller implemented as a LabVIEW application. A set of experiments were performed to test and optimize the sampling and image quality. We applied the DSA imaging protocol to record changes in the visualization of coronaries and myocardial perfusion induced by a vasodilator drug, nitroprusside. The drug was infused into a tail vein catheter using a peristaltic infusion pump at a rate of 0.07 mL/h for 3 min (dose: 0.0875 mg). Multiple DSA sequences were acquired before, during, and up to 25 min after drug infusion. Perfusion maps of the heart were generated in MATLAB to compare the drug effects over time. RESULTS: The best trade-off between the injection time, pressure, and image quality was achieved at 60 PSI, with the injection of 150 ms occurring early in diastole (60 ms delay) and resulting in the delivery of 113 μL of contrast agent. DSA images clearly show the main branches of the coronary arteries in an intact, beating heart. The drug test demonstrated that DSA can detect relative changes in coronary circulation via perfusion maps. CONCLUSIONS: The methodology for DSA imaging of rat coronary arteries can serve as a template for future translational studies to assist in safety evaluation of new pharmaceuticals. Although x-ray imaging involves radiation, the associated dose (0.4 Gy) is not a major limitation.