Nitric oxide (NOβ’), generated by nitric oxide synthase (NOS II) from immunostimulated cells during infection, plays an important role in host immune defense against microbial invasion. The impact of different rates of NOβ’ production on host cell function has not been defined. Herein, we describe the development of a method to express varied levels of murine NOS II in bovine pulmonary artery endothelial cells. A retroviral vector (pMFGSNOS) encoding NOS II was used to transduce primary cultures of endothelial cells. Bovine endothelial cells were susceptible to this transduction and up to 18% of the cells expressed immunodetectable murine NOS II. The NOS II-transduced endothelial cells were cultured on the three-dimensional matrix, Gelfoam, for 8 -10 days. Stable expression of NOS II was assessed by measuring nitrite accumulation in media every 2 days. By day 10, endothelial cells on Gelfoam were found to secrete NOβ’ at a rate exceeding 1.0 M/h/10 6 cells, concomi- tant with an enhanced level of NOS II activity. Argininosuccinate synthetase, a key enzyme in the metabolism of L-citrulline to L-arginine, increased as well, perhaps in response to dimunition of the intracellular arginine pool corresponding to the observed high output of NOβ’. In spite of the continuous flux of NOβ’, endothelial cell viability was not effected. This system provides the opportunity to assess the impact of dif-ferent levels of sustained NOβ’ production on endothelial cell physiology.