The three isoforms of nitric oxide synthase (NOS) are classified based on their mode of regulation by calmodulin (CaM). The "calcium-independent" inducible isoform (iNOS) contains tightly-bound CaM and is active at all levels of intracellular calcium. In contrast, the two calcium-dependent isoforms (neuronal, nNOS and endothelial eNOS) are activated by CaM-binding following stimulus-evoked increases in intracellular calcium. However, both the structural basis for these differences in regulation and the molecular basis for CaM-induced cNOS activation remained unclear. Given that the regulation of calmodulin regulated enzyme systems often involves the displacement of an intrinsic autoinhibitory domain, we attempted to identify regions of eNOS which may fulfill this autoinhibory function. Herein, I describe the identification of two autoinhibitory control elements (ACEs) in eNOS, one within the FMN binding domain (ACE-I) and one located at the C-terminus (ACE-2). Together with CaM, these form a tripartite system for the regulation of eNOS. ACE-lIACE-2 exerting negative effects to attenuate catalytic activity, which are overcome by the conformational changes induced by CaM-binding. The mechanism of ACE-lIACE-2-mediated inhibition and the alleviation of this inhibition were investigated.