Ca2+-independent forms of nitric-oxide synthase have significant activity when the endogenous calmodulin subunit is Ca2+ free, Further activation is seen when Ca2+ is added. We have examined the activation of a Ca2+-independent nitric-oxide synthase variant and its two point mutants that are more dependent on Ca2+ for activation using mutant calmodulins containing non-functional Ca2+-binding sites. These studies provide evidence that the Ca2+ independent activity of these enzymes can be exerted through specific adapted interactions between the enzyme and the Ca2+-binding site 2 of calmodulin, Further, the results suggest that EGTA-sensitive metals other than Ca2+ complexed to calmodulin may be involved in maximal activation of these nitric-oxide synthase variants.