Chinese hamster ovary cells were transfected with the human inducible nitric oxide synthase (iNOS) cDNA under the control of the hCMV promoter. The NOS inhibitor, S-ethyl isothiourea, was utilized to minimize toxicity from NO production. Selected colonies were grown in the absence of S-ethyl isothiourea, after which nitrite levels in the medium were measured. NOS activities in cell lysates were determined, and 9 colonies had iNOS activities of over 1 nmol of citrulline formed/min/mg protein. iNOS expression was further increased by gene amplification and the use of sodium butyrate, resulting in two cell lines with stable activity of greater than 3 nmol/min/mg. The iNOS purified from these cells had a specific activity of over 500 nmol/min/mg, and its properties were similar to native iNOS purified from cytokine-induced DLD-1 cells. This is the most efficient expression system reported to date for iNOS.