To better understand the structural factors that are important for a substrate to be oxidized by NOS with formation of NO, and to find new selective and efficient NO donors after in situ oxidation by NOS, we have synthesized a series of NOHA analogues and studied their NOS-catalyzed oxidation. We here report that, although the removal of the R-NH2 or the R-COOH groups of NOHA leads to a dramatic decrease of NO formation, removal of both groups leads to a simple non R-amino acid compound, N-butyl N′-hydroxyguanidine, which acts as a substrate almost as efficient as NOHA itself for NO production. Our results also show that it is so far the best NOS substrate among a series of N-alkyl N′-hydroxyguanidines.