The participation of nonmuscle myosin in force maintenance is controversial. Further, its regulation is difficult to examine in a cellular context as the light chains of smooth muscle and nonmuscle myosin co-migrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-LC) and nonmuscle myosin (NM-LC) were purified and a method was devised to resolve these proteins by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct LC isoelectric variants. These spots were identified as four distinct proteins; phosphorylated NM-LC (most acidic), non-phosphorylated NM-LC, phosphorylated SM-LC and non-phosphorylated SM-LC (most basic). During smooth muscle activation, NM-LC phosphorylation increased. During depolarization, the increase in NM-LC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II induced increase in NM-LC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous NMIIB knockout mice was significantly less than that of wild type littermates, suggesting that in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that in smooth muscle, the activation of NM myosin is regulated by Ca²⁺-calmodulin activated myosin light chain kinase during depolarization and a Rho kinase dependent pathway during agonist stimulation.