Objectives.-To determine the role of myosin light chain phosphorylation in feline colonic smooth muscle contraction. Sample Population.-Colonic tissue was obtained from eight 12- to 24-month-old cats. Procedure.-Colonic longitudinal smooth muscle strips were attached to isometric force transducers for measurements of isometric stress. Myosin light chain phosphorylation was determined by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stress and phosphorylation were determined following stimulation with ACh or SP, in the absence or presence of a calmodulin antagonist (W-7; 0.1 to 1.0 mM), myosin light chain kinase inhibitor (ML-9; 1 to 10 μM), or extracellular calcium - free solutions. Results.-Unstimulated longitudinal colonic smooth muscle contained low amounts (6.9 ± 3.2%) of phosphorylated myosin light chain. Phosphorylation of the myosin light chains was dose and time dependent with maximal values of 58.5% at 30 seconds of stimulation with 100 μM Ach and 60.2% at 45 seconds of stimulation with 100 nM SP. Active isometric stress development closely paralleled phosphorylation of the myosin light chains in ACh- or SP-stimulated muscle. W-7 and ML-9 dose dependently inhibited myosin light chain phosphorylation and isometric stress development associated with ACh or SP stimulation. Removal of extracellular calcium inhibited myosin light chain phosphorylation and isometric stress development in ACh-stimulated smooth muscle. Conclusions and Clinical Relevance.-Feline longitudinal colonic smooth muscle contraction is calcium-, calmodulin-, and myosin light chain kinase-dependent. Myosin light chain phosphorylation is necessary for the initiation of contraction in feline longitudinal colonic smooth muscle. These findings may prove useful in determining the biochemical and molecular defects that accompany feline colonic motility disorders.