Epithelial ion transport disorders including cystic fibrosis adversely affect male reproductive function by non-obstructive mechanisms and by obstruction of the distal duct. Continuous cell lines that could be used to define ion transport mechanisms in this tissue are not readily available. Porcine vas deferens epithelial cells were isolated by standard techniques and the cells spontaneously immortalized to form a cell line that is now titled PVD9902. Cells have been maintained in continuous culture for >4 years and 200 passages in a typical growth medium. Frozen stocks have been generated, and thawed cells exhibit growth characteristics indistinguishable from non-frozen counterparts. Molecular and immunocytochemical studies have confirmed the origin and epithelial nature of these cells. When seeded on permeable supports, PVD9902 cells grow as electrically tight (> 6000 cm²) confluent monolayers that respond to forskolin with an increase in short circuit current (I_sc; 8 ± 1 µAcm^-2) that requires Cl^-, HCO₃^-, and Na⁺, and is partially sensitive to bumetanide. mRNA is expressed for a number of anion transporters including CFTR, NBCe1b, DRA, pendrin, and CFEX. Both forskolin and isoproterenol cause an increase in cellular cAMP levels. In addition, PVD9902 cell monolayers respond to physiological (adenosine, norepinephrine) and pharmacological (NECA, isoproterenol) agonists with increases in I_sc. Unlike freshly isolated counterparts, however, PVD9902 cells do not respond to glucocorticoid exposure with an increase in amiloride-sensitive I_sc. RT-PCR analysis revealed the presence of both glucocorticoid and mineralocorticoid receptor mRNA as well as - and -ENaC, but not -ENaC. Nonetheless, PVD9902 cells recapitulate most observations made with freshly isolated cells and thus represent a powerful new tool to characterize mechanisms that contribute to male reproductive function.