The procedure of W. T. Perrie and S. V. Perry (1970, Biochem. J. 119, 31-38) has been improved and extended to allow a convenient large-scale isolation of the 20,000-Da light chain of vertebrate smooth muscle myosin. The method utilizes as source material tropomyosin-free actomyosin or myosin. The relatively pure light chain isolated from this material could be obtained in pure form by a single gel-filtration step. Separation of the unphosphorylated and phosphorylated light chain species was achieved by subsequent chromatography on a DEAE column. The solubility properties of this light chain, relevant to its use in myosin light chain kinase assays, were also established.