Cauda equina anatomy. I: Intrathecal nerve root organization. The three-dimensional organization of the human cauda equina has not been described previously. This is partly due to the difficulties of dissecting individual, unfixed nerve roots. By the use of a newly developed in situ fixation and embedding technique on 15 fresh human cadavers, the cross-sectional anatomy of the cauda equina was defined from L2-L3 to L5-S1. A highly consistent cross-sectional pattern was observed in all specimens. The lower sacral (S2-S5) and coccygeal roots were located in the dorsal aspect of the thecal sac, whereas the lumbar and first sacral roots exhibited an oblique, layered pattern as they ascended. The motor bundle was situated anteromedial to its respective sensory bundle within each layer, Invaginations of arachnoid held the nerve roots in a fixed relationship to one another. This previously undescribed three-dimensional anatomy within the thecal sac may aid in the understanding and treatment of trauma, neurocompressive syndromes, and tumors of the cauda equina. 