During the crawling movements of non-muscle cells, myosin II-containing structures assemble and disassemble with a high degree of spatial and temporal heterogeneity. In order to understand how this is controlled, we examined factors that influence the association of myosin II with detergent-resistant cytoskeletons of cultured endothelial cells. Treatment of cells with 0.05% Triton X-100 in an actin-stabilizing buffer released Ο³42% of the myosin II from the cytoplasm. Most remaining myosin II was dissociated from the cytoskeleton by treatment with ATP or AMPPNP, but not ADP, suggesting that myosin II is retained as ATP-sensitive filaments or via rigor-like binding to F-actin. Disruption of actin filaments with cytochalasin or latrunculin prior to detergent permeabilization sharply decreased the amount of myosin II retained, suggesting the latter type of association. Because phosphorylation of myosin II affects filament assembly and actin binding in vitro, phosphorylation levels in soluble and cytoskeletal myosin II were measured. Phosphorylation of myosin heavy chains was not significantly different between the two fractions, but regulatory light chains of cytoskeletal myosin II were 5 times more phosphorylated than in soluble myosin II. Tryptic-peptide mapping showed that cytoskeletal light chains were phosphorylated predominantly at serine 19/threonine 18, which regulates myosin II assembly in vitro, whereas soluble light chains were not phosphorylated or were phosphorylated at threonine 9. Treating cells with the kinase inhibitor, staurosporine, prior to permeabilization decreased light-chain phosphorylation with concomitant reduction in myosin retention. These observations suggest that assembly of myosin II into cytoskeletal structures, where it can generate and resist forces, is regulated in vivo by phosphorylation of myosin light chains at serine 19/threonine 18.