Aciculin is a recently identified 60-kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase type 1, (Belkin, A. M., Klimanskaya, I. V., Lukashev, M. E., Lilley, K., Critchley, D., and Koteliansky, V. E. (1994) J. Cell Sci. 107, 159-173). Aciculin expression in skeletal muscle is developmentally regulated, and this protein is particularly enriched at cell-matrix adherens junctions of muscle cells (Belkin, A. M., and Burridge, K. (1994) J. Cell Sci. 107, 1993-2003). The purpose of our study was to identify cytoskeletal protein(s) interacting with aciculin in various cell types. Using immunoprecipitation from cell lysates of metabolically labeled differentiating C2C12 muscle cells with anti-aciculin-specific antibodies, we detected a high molecular weight band (M(r) approximately 400,000), consistently coprecipitating with aciculin. We showed that this 400 kDa band comigrated with dystrophin and immunoblotted with anti-dystrophin antibodies. The association between aciculin and dystrophin in C2C12 cells was shown to resist Triton X-100 extraction and the majority of the complex could be extracted only in the presence of ionic detergents. In the reverse immunoprecipitation experiments, aciculin was detected in the precipitates with different anti-dystrophin antibodies. Immunodepletion experiments with lysates of metabolically labeled C2C12 myotubes showed that aciculin is a major dystrophin-associated protein in cultured skeletal muscle cells. Double immunostaining of differentiating and mature C2C12 myotubes with antibodies against aciculin and dystrophin revealed precise colocalization of these two cytoskeletal proteins throughout the process of myodifferentiation in culture. In skeletal muscle tissue, both proteins are concentrated at the sarcolemma and at myotendinous junctions. In contrast, utrophin, an autosomal homologue of dystrophin, was not codistributed with aciculin in muscle cell cultures and in skeletal muscle tissues. Analytical gel filtration experiments with purified aciculin and dystrophin showed interaction of these proteins in vitro, indicating that their association in skeletal muscle is due to direct binding. Whereas dystrophin was shown to be a major aciculin-associated protein in skeletal muscle, immunoblotting of anti-aciculin immunoprecipitates with antibodies against utrophin showed that aciculin is associated with utrophin in cultured A7r5 smooth muscle cells and REF52 fibroblasts. Immunodepletion experiments performed with lysates of metabolically labeled A7r5 cells demonstrated that aciculin is a major utrophin-binding protein in this cell type. Taken together, our data show that aciculin is a novel dystrophin- and utrophin-binding protein. Association of aciculin with dystrophin (utrophin) in various cell types might provide an additional cytoskeletal-matrix transmembrane link at sites where actin filaments terminate at the plasma membrane.