Activation of myosin light chain kinase (MLCK) and other kinases was studied in the arteries of transgenic mice that express an optical fluorescence resonance energy transfer (FRET) MLCK activity biosensor. Binding of Ca2+/calmodulin (Ca2+/CaM) induces an increase in MLCK activity and a change in FRET. After exposure to high external [K+], intracellular [Ca2+] (fura-2 ratio or fluo-4 fluorescence) and MLCK activity both increased rapidly to an initial peak and then declined, rapidly at first and then very slowly. After an initial peak (‘phasic’) force was constant or increased slowly (termed ‘tonic’ force). Inhibition of rho-kinase (Y-27632) decreased tonic force more than phasic, but had little effect on [Ca2+] and MLCK activation. Inhibition of PKCα and PKCβ with Gö6976 had no effect. KN-93, an inhibitor of CaMK II, markedly reduced force, MLCK FRET and [Ca2+]. Applied during tonic force, forskolin caused a rapid decrease in MLCK FRET ratio and force, but no change in Ca2+, suggesting a cAMP mediated decrease in affinity of MLCK for Ca2+/CaM. However, receptor (β-adrenergic) activated increases in cAMP during KCl were ineffective in causing relaxation, changes in [Ca2+], or MLCK FRET. At the same tonic force, MLCK FRET ratio activated by α1-adrenoceptors was ∼60% of that activated by KCl. In conclusion, MLCK activity of arterial smooth muscle during KCl-induced contraction is determined primarily by Ca2+/CaM. Rho-kinase is activated, by unknown mechanisms, and increases ‘Ca2+ sensitivity’ significantly. Forskolin mediated increases in cAMP, but not receptor mediated increases in cAMP cause a rapid decrease in the affinity of MLCK for Ca2+/CaM.