eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2-3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser602 lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr46 and Ser58 are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.