Both smooth and non-muscle class II myosin molecules are expressed in smooth muscle (SM) tissues comprising hollow organ systems. Individual smooth muscle cells may express one or more of multiple myosin II isoforms that differ in heavy and light chain subunits. Although much has been learned, the expression profiles, organization within contractile filaments, localization within cells, and precise roles in various contractile functions of these different myosin molecules are still not well understood. However, data supporting unique physiological roles for certain isoforms continues to build. Isoform differences located in the S1 head region of the myosin heavy chain (MHC) can alter actin binding and rates of ATP hydrolysis. Differences located in the MHC tail can alter formation, stability and size of the myosin thick filament. In these distinct ways, both head and tail isoform differences can alter force generation and muscle shortening velocities. The myosin light chains that are associated with the lever arm of the S1 head can affect the flexibility and range of motion of this domain, and possibly the motion of the S2 and motor domains. Phosphorylation of MLC20 has been associated with conformational changes in the S1 and/or S2 fragments regulating enzymatic activity of the entire myosin molecule. A challenge for the future will be delineation of the physiological significance of the heterogeneous expression of these isoforms in developmental, tissue, and species specific patterns, and or the intra- and intercellular heterogeneity of the myosin isoform expression in the SMCs of a given organ.