Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and alpha-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.