A quantitative and reproducible technique for establishing primary surface cultures from normal and diseased human muscle is described. Successful cultures were prepared from both fresh muscle and that stored up to 96 hr at 4 degrees C. The CPK activity of the muscle cells ranged between 0.5-3.0 micronmoles creatine per min per mg protein at 30 degrees C, thus indicating a high degree of differentiation. Spontaneous contractions were observed in 4 out of the 22 cultures established. Nerve cells were not required to achieve this level of differentiated function. No gross differences in plating efficiency, rate of myotube formation or CPK specific activity were found for the diseased muscle cells cultured so far. However, a 5--10-fold higher cell yield was obtained from muscles of patients with an inflammatory myopathy. The advantages of this technique for carrying out comparative studies on normal and dystrophic muscle cells are discussed.