L-NG-Nitroarginine (NA) inhibited both the L-arginine oxidation and the L-arginine-independent NADPH oxidation reactions catalyzed by the calcium/calmodulin-dependent constitutive nitric oxide synthase (cNOS) from bovine brain. NA binding did not require calmodulin, calcium, or NADPH. The onset of inhibition was slow with a second-order association rate constant (k(on) of 4.4 x 10(4) M-1 s-1. The dissociation rate constant (k(off) was 6.5 x 10(-4) s-1. The Kd value (k(off)/k(on)) of bovine brain cNOS for NA was 15 nM. L-Arginine was a competitive inhibitor of NA binding with a Ks value of 0.8 microM. The Km for L-arginine in the cNOS reaction was 1.2 microM. The NA binding sites of cNOS were titrated with NA, which enabled a kcat of 0.7 s-1, for the oxidation of L-arginine, to be calculated. Finally, a brain cNOS-(3H)NA complex was isolated. In contrast to the potent and slow onset of NA inhibition of brain cNOS, NA inhibition of inducible mouse macrophage NOS (iNOS) was weaker (Ki = 4.4 microM) and rapidly reversible. Thus, NA was a 300-fold more potent inhibitor of bovine brain cNOS than mouse macrophage iNOS.