Many advanced imaging application require the identification of seed and target ROIs. Due to size, and signal characteristics some brain structures including brainstem nuclei are difficult to identify with MR imaging, while other types of imaging such as PET have other drawbacks such as poor spatial resolution. We have come up with a “recipe” for defining the ventral tegmental area (VTA) as well as the dorsal raphe nucleus (DRN) using three anatomic atlases. The definition of these ROIs were confirmed with PET imaging. Our method allows for robust, reproducible ROIs using only a high resolution T1 weighted image.