Canavanine, an arginine analog, has been shown to be present to the extent of 15% in certain beans, such as Jack Beans. It is possible that canavanine could interfere with arginine metabolism. We have examined the effect of canavanine on brain nitric oxide synthase (NOS) which utilizes arginine as a substrate. Rat brain NOS has been transiently expressed in CHO cells by standard techniques. The effects of vaious inhibitors were examined using the expressed enzyme in order to understand the role of canavanine, as a possible neurotoxin. Canavanine inhibits the brain NOS causing a reduction in Vmax as well as an increase in the Km for arginine. A similar type of inhibition was obtained with other inhibitors of NOS, namely N(G)-methyl arginine and N(G)-nitro arginine. When 10 uM arginine was used as a substrate fifty percent inhibition of NOS was obtained with 0.3 muM nitro arginine, 5 muM N- methyl arginine and 15 ~M canavanine. Only the L-isomer of nitro arginine was a potent inhibitor. The D-isomer did not show any inhibition. Canaline, a metabolite of canavanine, did not inhibit NOS. Similar inhibition of NOS by canavanine and other NOS inhibitors was obtained with NOS isolated from rat cerebellum. These studies indicate that canavanine by inhibiting brain NOS may cause neurotoxic effects when human and animals are fed Jack Beans or other plant products containing canavanine.