Highlights ► MAP kinases bind to a pentabasic sequence in the eNOS autoinhibitory insertion. ► MAP kinases bind poorly or not at all to nNOS. ► MAP kinases interact weakly with CaM binding, increasing CaM release. ► The MAPK binding site is adjacent to the phosphorylation sites of other kinases. Endothelial nitric oxide synthase (eNOS) contains a motif similar to recognition sequences in known MAPK binding partners. In optical biosensing experiments, eNOS bound p38 and ERK with ∼100 nM affinity and complex kinetics. Binding is diffusion-limited (kon ∼ .15 × 106 M−1 s−1). Neuronal NOS also bound p38 but exhibited much slower and weaker binding. p38-eNOS binding was inhibited by calmodulin. Evidence for a ternary complex was found when eNOS bound p38 was exposed to CaM, increasing the apparent dissociation rate. These observations strongly suggest a direct role for MAPK in regulation of NOS with implications for signaling pathways including angiogenesis and control of vascular tone.