N omega-Propargyl-L-arginine (7) was synthesized as a potential mechanism-based inactivator of neuronal nitric oxide synthase (nNOS) and macrophage nitric oxide synthase (iNOS). Compound 7 is a potent reversible competitive inhibitor for both isoforms, having Ki values of 430 +/- 50 nM and 620 +/- 30 nM for nNOS and iNOS, respectively. These values are 12 and 32 times lower than the K(m) for L-arginine with nNOS and iNOS, respectively; however, 7 does not exhibit time-dependent inhibition with either. It also only undergoes oxidation very slowly. N omega-Hydroxy-N omega-propargyl-L-arginine also was synthesized to determine if the initial proposed enzyme-catalyzed hydroxylation of N omega-propargyl-L-arginine was problematic. This compound also is a potent reversible inhibitor of both nNOS and iNOS, but is not a time-dependent inactivator and is oxidized only very slowly. These results are in sharp contrast with the corresponding olefins, N omega-allyl-L-arginine and N omega-allyl-N omega-hydroxy-L-arginine recently reported to be potent time-dependent, irreversible inhibitors of nNOS (Zhang, H. Q.; Dixon, R. P.; Marletta, M. A.; Silverman, R. B., J. Am. Chem. Soc. 1997, 119, in press); N omega-allyl-L-arginine also was reported to be an inactivator of iNOS (Olken, N. M.; Marletta, M. A. J. Med. Chem. 1992, 35, 1137). This suggests that the active site of both isoforms of NOS can accommodate a variety of structures, but binding must have the appropriate juxtaposition for hydroxylation; otherwise, no oxidation occurs.