Five different proteolytic enzymes, trypsin, pronase, collagenase, papain and ficin, were tested for their ability to liberate mononucleated myogenic cells from adult rat muscle. Fragments of leg and thigh muscle from 350–400 gm rats were incubated with enzyme for one hour and mononucleated cells were separated from the partially digested fragments by centrifugation. Myogenic capacity of the isolated cells was tested in culture using procedures known to support differentiation of embryonic rat muscle. Although all the enzymes released viable cells from the muscle, myogenesis in vitro was obtained only from cells liberated by trypsin or pronase. The other three enzymes released fibroblasts, macrophages and fat cells but no presumptive myoblasts. Light and electron microscopic examination of the digested muscle revealed that the enzymatic release of myogenic cells is dependent upon removal of the basement lamina surrounding the fibers. Trypsin and pronase dissolve the basement lamina, whereas collagenase, papain or ficin do not. These findings demonstrate that a population of myogenic stem cells exists in fully mature, non-regenerating adult muscle. The frequency of these cells and the conditions required for their release suggest that they are identical with the muscle satellite cells.