Pressure is an important physiological regulator, but under abnormal conditions may be a critical factor in the onset and progression of disease in many organs. In vivo, proximal tubular epithelial cells are subjected to pressure as a result of ureteral obstruction, which may influence the production of nitric oxide (NO), a ubiquitous multi-functional cytokine. To directly explore the effect of pressure on the expression and activity of nitric oxide synthase (NOS) in cultured proximal tubular epithelial cells, a novel pressure apparatus was developed. Cells were subjected to pressures of 20- 120 mmHg over time (5 min- 72hrs). Reverse transcriptase PCR (RT-PCR) demonstrated an increase in iNOS and sGC, while eNOS remained unchanged. Real time-PCR (qPCR) confirmed an earlier induction of iNOS transcript subjected to 60 mmHg as compared to cytokine mix (CM). iNOS protein expression was significantly increased following 60 mmHg of pressure for 24 hrs. Use of nuclear factor kappa B (NF-kB) inhibitors was shown to prevent the increase in iNOS expression following 60mmHg for 2hrs. NO and cGMP were increased with the application of pressure. Addition of the irreversible iNOS inhibitor (1400W) was shown to prevent this increase. We demonstrate that with the use of a simply designed apparatus, pressure led to an extremely early induction of iNOS and a rapid activation of NOS activity to increase NO and cGMP in proximal tubule epithelial cells. The rapid effects of pressure on iNOS may have important implications in the obstructed kidney.