Calmodulin binding to inducible nitric oxide synthase may play an important role in its Ca2+-independent activity. Studies of inducible nitric oxide synthase chimeras containing the calmodulin-binding sequence of neuronal or endothelial nitric oxide synthases show that the calmodulin-binding sequence of inducible nitric oxide synthase is necessary but not sufficient for the Ca2+-independent activity. The mutations at lysine 525 located at the C-terminus of the calmodulin-binding sequence of inducible nitric oxide synthase were examined for the effects on the Ca2+-independent activity with chimeras containing the oxygenase or reductase domains of inducible or neuronal nitric oxide synthases. Results show that the Ca2+-independent binding of calmodulin is not solely responsible for maximal Ca2+-independent activity of inducible nitric oxide synthase. Lysine 525 of inducible nitric oxide synthase may also play an important role in coordinating the maximal Ca2+-independent activity.