C₃-tris-malonyl-C₆₀-fullerene and D₃-tris-malonyl-C₆₀-fullerene derivatives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution. The inhibition of citrulline formation by C₃-tris-malonyl-C₆₀-fullerene occurs with IC₅₀ values of 24, 17, and 123 μM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 μM l -arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 μM C₃-tris-malonyl-C₆₀-fullerene. This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0. C₃-tris-malonyl-C₆₀-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC₅₀ value of 22 μM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 μM. The inhibition of nNOS activity by C₃-tris-malonyl-C₆₀-fullerene reduced the maximal velocity of product formation but did not alter the EC₅₀ value for activation by calmodulin. C₃-tris-malonyl-C₆₀-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the K_m for l -arginine substrate or the EC₅₀ value for tetrahydrobiopterin cofactor. As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C₃-tris-malonyl-C₆₀-fullerene did not produce a dissociation of nNOS dimers into monomers. These observations are consistent with the proposal that C₃-tris-malonyl-C₆₀-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface.