The morphologic characteristics of the medullary and cortical segments of the ascending thick limb of Henle were examined in the rat utilizing light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The kidneys of six normal female Sprague-Dawley rats were preserved for morphological examination utilizing in vivo intravascular perfusion with 6.25% glutaraldehyde buffered in 0.1m sodium cacodylate. SEM revealed the presence of two distinctly different patterns of surface structure among the population of cells forming the epithelium of the ascending thick limb, a roughsurfaced cell with prominent microvilli (R cell) and a smoothsurfaced cell generally devoid of extensive microvilli except along the lateral cell margins (S cell). This variation in surface structure was confirmed with light microscopy and TEM. Throughout both the medullary and cortical segments of the ascending thick limb, the R cells possessed extensive lateral processes radiating form the main cell body to interdigitate with adjacent cells. Lateral processes of S cells were less common than those of the R cells in the inner stripe of the outer medulla, but increased in number and complexity in the outer stripe of the outer medulla and throughout the cortex. S cells outnumbered R cells in the inner stripe of the outer medulla. As the thick limb transversed the outer medulla and ascended toward the parent renal corpuscle, there was a marked increase in the relative number of R cells. In the cortical segment of the ascending thick limb, both the R and S cells tended to acquire a more extensive population of microvilli over their luminal surface making it increasingly difficult to establish clearcut distinctions between the two surface patterns. With the possible exception that vesicles and tubulovesicular profiles located in the apical region were more numerous in many of the S cells, additional morphological differences between the rough and smoothsurfaced cells were not evident. Whether the morphological results represent two distinct cell types or simply a variation in the structure of a single cell type, their presence must be considered in future structural-functional studies involving the ascending thick limb of Henle in the rat.