Much of our knowledge of brain function has been gleaned from studies using microelectrodes to characterize the response properties of individual neurons in vivo. However, because it is difficult to accurately determine the location of a microelectrode tip within the brain, it is impossible to systematically map the fine three-dimensional spatial organization of many brain areas, especially in deep structures. Here, we present a practical method based on digital stereo microfocal x-ray imaging that makes it possible to estimate the 3D position of each and every microelectrode recording site in "real time" during experimental sessions. We determined the system's ex vivo localization accuracy to be better than 50 µm, and we show how we have used this method to co-register hundreds of deep-brain microelectrode recordings in monkeys to a common frame of reference with median error of less than 150 µm. We further show how we can co-register those sites with magnetic resonance (MR) images, allowing for comparison with anatomy, and laying the groundwork for more detailed electrophysiology/fMRI comparison. Minimally, this method allows one to marry the single-cell specificity of microelectrode recording with the spatial mapping abilities of imaging techniques, and it has the potential of yielding fundamentally new kinds of high-resolution maps of brain function.