Abstract: The present invention is directed to overproduction of nitric oxide synthase (NOS) in a prokaryote. More particularly, the invention involves overexpression of functional neuronal NOS in a protease-deficient strain of Escherichia coli, utilizing a pCW vector under the control of the tac promotor. The invention further involves co-expression of the protein with folding agonists, or chaperonins. The enzyme produced is complete with heme and flavins, and may be activated by incubation with tetrahydrobiopterin. It may be isolated as a predominantly high spin heme protein that demonstrates spectral properties which are identical to those of nNOS isolated from human kidney 293 cells. The methods disclosed are contemplated to be useful in expressing large amounts of other nitric oxide synthases, as well as other proteins that are difficult to produce correctly folded in prokaryotes.