Actin and myosin or actomyosin filaments also play a significant role in cells other than those involved in muscle contraction (which occurs within the sarcomere of muscle cells). The mechanism of non-muscle cell contractile bundles was first observed in Dictyostelium and Acanthamoeba. In non-muscle cells, two bundles are commonly found: stress fibers and actomyosin adherence belts. These contractile bundles are smaller and less organized than the ones found in muscle cells. They are held together by accessory proteins such as fascin, filamin, and fimbrin depending on where they are located. The formation and contractile ability of these actomyosin bundles are regulated by phosphorylation.

In non-muscle cells, actin and non-muscle myosin II (NMM) filaments combine to form stress fibers. There are about 15 to 20 myosin filaments in each bundle. The mechanism of action of these actinomyosin contractile bundles is similar to those in muscle fibers, where ATP hydrolysis by the myosin globular head drives the actin contraction. The stress fibers, along with the focal adhesions present at the cell edges, regulate the mechanosensitive machinery in the cells. These bundles are also responsible for regulating cell morphogenesis. In cells, these stress fibers are found near the cell edges, where they help the cell anchor on the substratum.

In epithelial cells, the actomyosin bundles are attached to the adhesion junctions as circumferential or adherence belts close to the plasma membrane. These belts regulate the apical constriction of polarized epithelial cells. In dividing cells, these actomyosin bundles are recruited by the septin ring at the cell cleavage furrow to form the contractile ring, which is a major step in cytokinesis.