The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC(20)) at Ser(19) by Ca(2+)/calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca(2+)-independent phosphorylation at both Ser(19) and Thr(18) by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr(18) on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser(19) and Thr(18) was achieved by treatment with adenosine 5'-O-(3-thiotriphosphate) in the presence of Ca(2+), which induced stoichiometric thiophosphorylation at Ser(19), followed by microcystin (phosphatase inhibitor) in the absence of Ca(2+), which induced phosphorylation at Thr(18). Phosphorylation at Thr(18) had no effect on steady-state force induced by Ser(19) thiophosphorylation. However, phosphorylation of Ser(19) or both Ser(19) and Thr(18) to comparable stoichiometries (0.5 mol of P(i)/mol of LC(20)) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: t(½) values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser(19)) and diphosphorylated LC(20), respectively. We conclude that phosphorylation at Thr(18) decreases the rates of LC(20) dephosphorylation and smooth muscle relaxation compared with LC(20) phosphorylated exclusively at Ser(19). These effects of LC(20) diphosphorylation, combined with increased Ser(19) phosphorylation (Ca(2+)-independent), may underlie the hypercontractility that is observed in response to certain physiological contractile stimuli, and under pathological conditions such as cerebral and coronary arterial vasospasm, intimal hyperplasia, and hypertension.