Using interferometry–based biosensors the binding and release of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion limited and within approximately three orders of magnitude of the Smoluchowski limit imposed by orientation–independent collisions. This suggests that the orientation of CaM is facilitated by the charge arrays on the CaM–binding site and the complementary surface on CaM. Protein kinase C phosphorylation of eNOS T495, adjacent to the CaM–binding site, abolishes or greatly slows CaM binding. Kinases which increase the activity of eNOS did not stimulate the binding of CaM, which is already diffusion limited. The coupling of Ca2+ binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca2+ dependence of CaM binding. Hence, changes in the Ca2+ sensitivity of CaM binding always imply changes in the NOS–CaM affinity. It is possible, however, that in some regimes binding and activation are not synonymous, so that Ca2+ sensitivity need not be tightly linked to CaM sensitivity of activation. This study is being extended using mutants to probe the roles of individual structural elements in binding and release.