A new technique for the sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation of rat skeletal muscle myosin heavy-chain (MHC) isoforms is presented. This technique allows for the separation of the four identified MHC isoforms known to be present in adult rat skeletal muscle. These types of MHC are commonly called I, IIa, IIx or IId, and IIb. The procedure can be performed using minigel electrophoresis systems and does not involve preparation of gradient-separating gels or the use of special cooling devices. The procedure accommodates both silver and Coomasie Blue staining. Thus the procedure is simple to perform and highly repeatable, providing high-resolution separation of MHC protein isoforms. The percent composition of the four adult MHCs in rat soleus, medial gastrocnemius, diaphragm, and levator ani muscles by use of this procedure and Coomasie Blue staining is similar to that previously reported. This new technique provides a novel and easy-to-perform method for the separation of rat skeletal muscle MHC isoforms.