The Acridine Orange (AO) stain for muscle biopsies is particularly useful to identify regenerating and ongoing hypertrophic muscle fibers under fluorescent microscopy. This method was applied to muscle biopsies from 65 patients who suffered from various childhood neuromuscular disorders. While normal fibers showed dull green cytoplasm with small green-yellow nuclei, striking fluorescent fibers were observed in eight cases of congenital muscular dystrophy (CMD) and 12 cases of Duchenne muscular dystrophy (DMD); these fibers were characterized as follows: (1) small fibers with big oval or spherical nuclei which fluoresced strongly with a bright orange color; (2) fibers of various sizes and different degrees of orange fluorescence; and (3) opaque fibers with bright yellow cytoplasm. The small diameter fibers in Werdnig-Hoffmann (WH) disease, nemaline myopathy, and congenital fiber type disproportion failed to show apparent AO-RNA fluorescence. Although all the atrophic fibers in Kugelberg-Welander (KW) disease showed a vague orange fluorescent color, this was obviously different from that of regenerating fibers seen in CMD and DMD. In addition to these findings, the hypertrophic fibers in a case of unclassified myopathy also showed moderate orange fluorescence around the entire periphery of the cytoplasm.