Reduced nitric oxide (NO) bioavailability and impaired vascular function are the key pathological characteristics of inflammatory diseases such as atherosclerosis. We have recently found that leukocyte-derived hypochlorous acid is able to react with the nitric-oxide synthase (NOS) substrate L-arginine to produce chlorinated L-arginine (cl-L-Arg). Interestingly, cl-L-Arg potently inhibits the formation of NO metabolites in cultured endothelial cells. It is unknown whether cl-L-Arg has a direct inhibitory effect on endothelial NOS (eNOS). In addition, the effect of cl-L-Arg on the other NOS isoforms, neuronal NOS (nNOS) and inducible NOS (iNOS), is also unknown. Therefore, we designed the current study to test the effects of cl-L-Arg on eNOS, nNOS, and iNOS. Using recombinant NOS, we found that cl-L-Arg had a direct inhibitory effect on the activity of NOS. The effect of cl-L-Arg on NOS activity is nonselective, as all three NOS isoforms were inhibited with a similar IC(50). We further determined the effect of cl-L-Arg on the three NOS isoforms at the tissue level. The results demonstrated that cl-L-Arg potently inhibited all three NOS isoform-mediated vessel reactivities, as well as the NOS signaling molecule cGMP. Cl-L-Arg might serve as a novel endogenous NOS inhibitor and an important mediator for vascular dysfunction under inflammatory conditions such as atherosclerosis. Blocking cl-L-Arg formation may be a new therapeutic approach to cardiovascular diseases.