Prostasin is a glycosylphosphatidylinositol anchored serine protease necessary for acquisition of barrier function of epidermis (Leyvraz et al. 2005). Prostasin is expressed in adult kidney. In rat, urine concentrating ability develops in postnatal weeks 2–3 governed by glucocorticoid. We hypothesized that prostasin is sensitive to glucocorticoid and essential for tight junction formation in renal collecting duct. The mouse cortical collecting duct cell line M-1 was used to study formation of tight epithelium in vitro. M-1 cells grew on semipermeable membranes which allowed measurements of transepithelial resistance (TER) and –voltage (TEV). Cells were seeded at 4x10⁵/ml and developed a significant increase in TER (79.8 ohm 3.4 to 1346 ohm 213, p=0.0001, n=6) and TEV (1.55mV0.2 to 20.7mV3.8, p=0.0005, n=6). In the same period prostasin mRNA and protein level increased significantly in M-1 cells. Omission of the synthetic glucocorticoid dexamethasone from the culture medium abolished the increase in prostasin mRNA and impaired the development of TER (1346 ohm 212.9 vs. 403.3 ohm 44.7, p=0.0015, n=6) and TEV (20.7mV3.8 vs. 0.6 mV0.2, p=0.0003, n=6). Addition of the serine protease inhibitor, aprotinin, to the apical but not the basolateral side of M-1 monolayers inhibited the development of TER (1497 ohm 183 vs. 997.2 ohm 131, p=0.04, n=12) and TEV (19.1mV1.5 vs. 10.2mV1.7, p=0.0008, n=12) relative to the controls. In a developmental series of rat kidneys, prostasin mRNA abundance increased significantly between postnatal days 0–20, when urine concentrating capacity increases ~5 times. Serine protease activity and glucocorticoids are necessary for development of tight collecting duct epithelium in vitro. Prostasin is a candidate protease that displays sensitivity to glucocorticoids. Leyvraz, C. et al. 2005. J. Cell Biol., 170, 487–496.