Epididymal epithelium is well known as a site of secretion of various proteins present in epididymal luminal fluid. Although there have been many reports of primary cultures of epididymal epithelial cells, their growth is limited over time. We have established immortalized epididymal epithelial cell lines from primary cultures of epididymal cells from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene in order to study the regulatory mechanisms of epididymal function, including specific factor secretion. These cell lines (PC1 from proximal caput; and DC1, DC2, and DC3 from distal caput) have been maintained for more than 1 year and show temperature-dependent growth and expression of cytokeratin, a marker of epithelial cells. These cells express the androgen receptor as well as markers of the murine epididymal epithelium, PEB-like protein (ie, phosphatidye ethanolamine binding protein), E-RABP (ie, epididymal retinoic acid-binding protein), and EP17 (ie, epididymal protein of 17 kd). The androgen-regulated 5-kilobase mE-RABP promoter DNA fragment ligated to the neomycin-resistant gene was used for stable transfection of DC1 cells. Because the mE-RABP gene is specifically expressed in the distal caput, neomycin selection provides a pure population of epithelial cells from that segment. This neomycin-resistant immortalized cell line from the distal caput was cultured for more than 6 months. Such immortalized cell lines should be valuable tools for studying the regulation of tissue-specific gene expression, and may be used to identify one or more epididymal specific transcription factors involved in the expression of epididymal specific proteins.