OBJECTIVES/HYPOTHESIS: The purpose of the present study is to describe in detail the ultrastructure of the human endolymphatic sac using a new and improved method of fixation as well as a refined surgical approach in obtaining specimens. STUDY DESIGN: Transmission electron microscopy of the human endolymphatic sac, employing an oxygenated fixative. METHODS: Eighteen tissue samples of the human endolymphatic sac were obtained during surgery for vestibular schwannoma using the translabyrinthine approach. The specimens were fixed in 2% glutaraldehyde in an oxygenated fluorocarbon blood substitute vehicle before preparation by routine methods for transmission electron microscopy. We focused on the epithelial cell layer, subepithelial tissue, intraluminal content, and vascular tissue in both the intra- and extraosseous part of the endolymphatic sac. RESULTS: We observed well-defined endolymphatic sac epithelial cell lining in all 18 specimens. In general, we found very well-preserved specimens with well-defined intracellular structures. In contrast to the results in former studies, a minimum of fixation artifacts was observed in the present study. Three different cell types were observed in the intraosseous part of the sac: mitochondria-rich cells, ribosome-rich cells, and nonclassifiable cells. A fourth cell type was found in the extraosseous part. Novel ultrastructural features of the epithelial lining and the subepithelial layer are described and discussed. CONCLUSIONS: The results in the present study indicate an improvement in obtaining human tissue with optimal fixation for ultrastructural analysis and provide several novel morphologic observations. The potential functions of the endolymphatic sac are discussed with reference to former studies.