Multilayer cell clusters have been observed before confluence and before myotube formation in muscle cell cultures derived from open biopsies of 7 of 14 (50%) female carriers of Duchenne muscular dystrophy, and in a high percentage of other dystrophic cultures. By contrast, this abnormality was seen in only 12 of 204 (6%) muscle biopsies from patients with other neuromuscular disorders. It appears that cluster formation is independent of the amount of connective tissue present in vivo, because histopathological analysis of the carrier biopsies showed increased endomysial connective tissue in only two cases. These results suggest that cluster formation is an expression of a myogenic defect and that it may be a manifestation of the genetic abnormality in X-linked muscular dystrophy.