<i>Background:</i> An understanding of the relationships between the anterior commissure-posterior commissure line (AC-PC), the subthalamic nucleus (STN), and red nucleus (RN) is imperative if these structures are to be used for targeting in deep brain stimulation. Currently, these relationships are incompletely understood and difficult to assess using conventional MRI. We examined the location and relationships of the STN and the RN to the AC-PC line and to each other in order to provide a greater understanding of their utility when targeting the STN, and the consistency of these anatomic relationships when examined using conventional MRI. <i>Methods:</i> A total of 52 STN and RN in 26 patients with Parkinson’s disease were evaluated on T₂-weighted MR images. The anterior and posterior commissures and the border coordinates of the STN and RN were derived using frame coordinates. The distances from the midcommissural point (mcp) to the centers of the STN and RN, the diameters for each nucleus, and the distances between the nuclei were calculated in the x-, y-, and z-axes. <i>Results:</i> The mean AC-PC length was 26.1 ± 1.3 mm. The distance from the mcp to the center of the STN was 10 ± 0.7 mm in the x-axis, 0.2 ± 0.7 mm in the y-axis, and 3.3 ± 0.9 mm in the z-axis. The distance from the mcp to the center of the RN was 4.7 ± 0.6 mm in the x-axis, –5.9 ± 1.0 mm in the y-axis, and 6.1 ± 1.3 mm in the z-axis. The distance between the STN and RN was 2.3 ± 0.7 mm in the x-axis, 2.1 ± 1.0 mm in the y-axis, and –0.2 ± 1.3 mm in the z-axis. <i>Conclusions:</i> Although recent studies imply that the RN can be used as a relatively consistent marker for the position of the STN, the present data suggest otherwise. These data indicate that a single targeting method may be inadequate given the resolution of conventional MRI, and that it is imperative to use multiple anatomical measurements when targeting the STN for deep brain stimulation in Parkinson’s disease.