Background The anatomy of the substantia nigra on conventional MRI is controversial. Even using histological techniques it is difficult to delineate with certainty from surrounding structures. We sought to define the anatomy of the SN using high field spin-echo MRI of pathological material in which we could study the anatomy in detail to corroborate our MRI findings in controls and Parkinson's disease and progressive supranuclear palsy. Methods 23 brains were selected from the Queen Square Brain Bank (10 controls, 8 progressive supranuclear palsy, 5 Parkinson's disease) and imaged using high field 9.4 Tesla spin-echo MRI. Subsequently brains were cut and stained with Luxol fast blue, Perls stain, and immunohistochemistry for substance P and calbindin. Once the anatomy was defined on histology the dimensions and volume of the substantia nigra were determined on high field magnetic resonance images. Results The anterior border of the substantia nigra was defined by the crus cerebri. In the medial half it was less distinct due to the deposition of iron and the interdigitation of white matter and the substantia nigra. The posterior border was flanked by white matter bridging the red nucleus and substantia nigra and seen as hypointense on spin-echo magnetic resonance images. Within the substantia nigra high signal structures corresponded to confirmed nigrosomes. These were still evident in Parkinson's disease but not in progressive supranuclear palsy. The volume and dimensions of the substantia nigra were similar in Parkinson's disease and controls, but reduced in progressive supranuclear palsy. Conclusions We present a histologically validated anatomical description of the substantia nigra on high field spin-echo high resolution magnetic resonance images and were able to delineate all five nigrosomes. In accordance with the pathological literature we did not observe changes in the nigrosome structure as manifest by volume or signal characteristics within the substantia nigra in Parkinson's disease whereas in progressive supranuclear palsy there was microarchitectural destruction.