We propose the use of Tomographic Digital Subtraction Angiography (TDSA) for functional imaging in small animals. TDSA combines the advantages of high temporal resolution of digital subtraction angiography (DSA) and high spatial resolution of micro-computed tomography (CT). TDSA augments projection imaging methods such as DSA by providing three-dimensional information using tomosynthesis or CT reconstruction algorithms. Thus, four-dimensional (4D) datasets with a temporal resolution on the order of 100 ms and spatial resolution ranging from 100 microns to 1 mm, depending on the scanning angle, can be obtained. The approach is based on the novel paradigm that the same time density curves can be reproduced in a number of consecutive injections of microL volumes of contrast at a series of different angles of rotation. © 2006 IEEE.