Phosphorylation of the 20 000-Da light chain of myosin in functionally different porcine carotid arteries was determined, with use of two-dimensional gel electrophoresis. Stretching arteries to 1.7 times their resting length resulted in maximal phosphorylation. Intracellular Ca2+ was mobilized for stretch-induced light chain phosphorylation. Releasing the stretch from the arteries produced active tension spontaneously, without the participation of any exogenous stimulating agent. Prolonged treatment of arteries with a chelating agent (EGTA) not only abolished stretch-induced phosphorylation, it also prevented the development of active tension when the stretch was released. However, when the EGTA was washed out and the strips were restretched and again released, the stretch-induced phosphorylation and the stretch-release-induced active tension were restored. Evidently, arteries must contain phosphorylated light chain if they are to produce active tension. The myosin light chain became partly dephosphorylated in arteries that developed active tension when the stretch was released, but more than half of the light chains remained phosphorylated. This result suggests that phosphorylation of light chain is involved, not in the generation of active tension, but rather in activation of smooth muscle.