We demonstrate here that neuronal nitric-oxide synthase (nNOS) is phosphorylated and inhibited by a constitutively active form of Ca 2 + /calmodulin (CaM)-dependent protein kinase I (CaM-K I1-293). Substitution of Ser 7 4 1 to Ala in nNOS blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser 7 4 1 by Ser to Asp mutation resulted in decreased binding of and activation by CaM, since the mutation was within the CaM-binding domain. CaM-K I1-293 gave phosphorylation of nNOS at Ser 7 4 1 in transfected cells, resulting in 60-70% inhibition of nNOS activity. Wild-type CaM-K I also did phosphorylate nNOS at Ser 7 4 1 in transfected cells, but either CaM-K II or CaM-K IV did not. These results raise the possibility of a novel cross-talk between nNOS and CaM-K I through the phosphorylation of Ser 7 4 1 on nNOS.