TRKE-1 is a pure line of epithelium-like neoplastic cells derived from the kidney of a rat treated 48 hr previously with a carcinogenic dose of dimethylnitrosamine. Using light microscopy, the line was characterized by cohesive growth behavior typical for epithelium and the formation of hemicysts (domes) at postconfluence. Enhancement of dome formation by dibutyryl cyclic adenosine 3':5'-monophosphate and dimethyl sulfoxide and inhibition by ouabain established these structures as a manifestation of differentiated cellular function, namely, transepithelial fluid transport. Structurally, TRKE-1 cells in monolayer culture were characterized by apical distribution of microvilli, cilia, and endocytic vesicles, ordered sequence of junctional components at the apical lateral border including tight junction and desmosomes, basolateral cellular interdigitations below the junctional complex, basal location of microfilament bundles, and a conspicuous content of mitochondria. Each of these features typifies mammalian renal tubule epithelium in vivo. The occasional profusion of microvilli; the prominent, apically distributed endocytic vesicles; and the well-developed basal microfilament tracts suggest, in particular, that the proximal segment of the nephron may represent the site of origin of this transformed cell line. The various morphological aspects of renal epithelial differentiation were also expressed in multicellular tumor spheroids grown in suspension, with an accentuation of junctional complexes, endocytic vesicles, and intracytoplasmic lumina. In addition, this three-dimensional culture mode supported cellular organization into acinar profiles suggestive of primitive tubule formation. In confirming the epithelial nature of TRKE-1 and a possible identity with the proximal tubule, this study provides an in vitro animal model representative of chemically transformed renal epithelium which may be analogous to human renal cell carcinoma.