Objective The formation of the osmotic gradient in the renal medulla has close relation with epithelial transport and the spatial arrangement of Henle,s loops and vessels.The objective of the present study was to investigate the tubular course of the long loop nephrons (LLN) in the medulla. Methods Three C57BL/6J mice were fixed by perfusion and embedded in Epon 812.Tissue blocks were cut perpendicularly to the longitudinal axis of the kidney,and a total of 2 000serial sections with a thickness of 2.5 μm were obtained from the surface of the kidney to the inner medulla.Using image recording and alignment, 26 LLN were identified,traced and 3D-reconstructed with a series of computer programs written in the C programming language. Results The LLN initiated at the mid-cortex and juxtamedullary cortex of the kidney.Their bends were located at various levels of theinner medulla,but the deepest bends reached the papillary.Interestingly,descending thin limbs of the LLN ran tortuously in the outer part of the inner stripe of the outer medulla,and the length of the tortuous part of the tubules was two times of the direct distance in that area.The winding descending thin limb ran in distance but paralleled with its isogenous thick ascending limb.The thick ascending limbs of the longer LLN ran in the vascular bundles in the inner stripe of the outer medulla.In the inner part of the inner stripe of the outer medulla,descending thin limbs ran adjacently with thick ascending limbs. Conclusion A winding course of descending thin limbs in the inner stripe of the outer medulla of LLN increases in the length of the corresponding segments,suggesting an impact in the formation of the osmotic gradie