The 20-kD regulatory light chain (RLC) plays a central role in the regulation of smooth muscle contraction. Little is known about the structure or expression of smooth muscle myosin light chain (MLC) genes. A cDNA library was constructed in the expression vector, lambda gt-11, with mRNA derived from cultured rat aortic smooth muscle cells. Using antibody generated against tracheal smooth muscle myosin, three cDNA clones encoding a RLC were isolated, one of which, SmRLC-2, represents a full-length transcript of the RLC mRNA. The derived amino acid sequence shows 94.2% homology with the chicken gizzard RLC, and 70 and 52% homology with the rat skeletal and cardiac muscle MLC-2 proteins, respectively. Thus, the gene encoding the putative smooth muscle RLC appears to have originated by duplication of the same ancestor that gave rise to the sarcomeric MLC-2 genes. Contrary to the stringent tissue-specific expression of sarcomeric MLC-2 genes, RNA blot hybridization and S1 nuclease mapping demonstrates that the putative smooth muscle RLC gene is expressed in smooth, sarcomeric, and nonmuscle tissues at significant levels. Primer extension analysis suggests that the same promoter region is used in these different tissues. Thus the putative smooth muscle RLC gene appears to be a gene that is constitutively expressed in a large variety of cells and has a differentiated function in smooth muscle.