Three modified procedures for the separation of cardiac myosin light chains are carefully compared. Ion-exchange chromatography gives a purified cardiac myosin light chain 1, whereas light chain 2 is always contaminated by light chain 1. Reversed-phase high-performance liquid chromatography gives the best resolution of these light chains and needs only 20 min for each run. However, it requires pure preparation of myosin light chains before separation. Isoelectric precipitation is the simplest procedure and suitable for large quantities of material. Although it gives the highest yield the separation is not adequate. A modified and rapid procedure for the isolation of cardiac and skeletal total myosin light chains is also presented.