A cloned cell line that spontaneously polarizes in standard glucose-containing media was derived from a single cell of the adenocarcinoma cell line HT-29. The cloned line, designated HT-29/cl.f8, has remained stable over 2 yr in culture, maintained high transepithelial resistance (300 ohm cm² or higher), and correctly sorted influenza virus and vesicular stomatitis virus to apical or basolateral domains, respectively. The newly cloned cells also displayed apical microvilli, tight junctions, and desmosomes, the morphological characteristics of mature epithelia. The cloned HT-29/cl.f8 cells function as epithelial enterocytes as shown by the apical expression of intestinal alkaline phosphatase, the expression of vimentin and cytokeratin, and lack of expression of mucin. We propose that the newly cloned HT-29/cl.f8 cells offer a viable alternative for studies of enterocyte function that will readily yield interpretable data not complicated by cell alterations due to the presence of drugs or chemicals that induce differentiation.