The availability of genetically altered animal models of human disease for basic research has generated great interest in new imaging methodologies. Digital subtraction angiography (DSA) offers an appealing approach to functional imaging in small animals because of the high spatial and temporal resolution, and the ability to visualize and measure blood flow. The micro-injector described here meets crucial performance parameters to ensure optimal vessel enhancement without significantly increasing the total blood volume or producing overlap of enhanced structures. The micro-injector can inject small, reproducible volumes of contrast agent at high flow rates with computer-controlled timing synchronized to cardiopulmonary activity. Iterative bench-top and live animal experiments with both rat and mouse have been conducted to evaluate the performance of this computer-controlled micro-injector, a first demonstration of a new device designed explicitly for the unique requirements of DSA in small animals. Injection protocols were optimized and screened for potential physiological impact. For the optimized protocols, we found that changes in the time-density curves for representative regions of interest in the thorax were due primarily to physiological changes, independent of micro-injector parameters.