OBJECT: The conus medullaris and the nerve roots from S-1 to S-5 regulate bladder function as well as movement and sensation of the lower extremities. This most caudal region of the spinal cord has not been studied in great detail anatomically despite its important regulatory role. The goal of this analysis is to characterize the normal intradural microanatomy of the sacral nerve roots at their origin from the conus medullaris. METHODS: The thecal sacs from 20 cadavers were fixated in formaldehyde and dissected under the operative microscope. RESULTS: More than 50 rootlets originated from the conus medullaris over a distance of < 3 cm. The rootlets were loosely organized into bundles by the arachnoid membrane with decreasing diameters. These diameters were 1.7 mm (ventral)/2.4 mm (dorsal) at S-1, and 0.17 mm (ventral)/0.4 mm (dorsal) at S-5. The roots were separated by neither the dentate ligament nor interradicular gaps. The number of rootlets decreased in the rostrocaudal direction with 2 ventral and 5 dorsal rootlets at S-1, but only 1 ventral (inconsistently found) and 2 dorsal rootlets at S-5. Typically, 1 nerve anastomosis was present between adjacent dorsal roots from S-1 to S-4. Nerve anastomoses between ventral roots or rootlets of the same root were less frequent. The dorsal segment of origin (linea radicularis) decreased in length from 7.2 mm at S-1 to 4.8 mm at S-5. CONCLUSIONS: The current study provides anatomical details and specific morphometric data of the intradural contents at the level of the conus medullaris. This information is valuable for intraoperative orientation, endoscopic navigation, and possible intradural nerve stimulation.