The dystrophin-associated protein complex (DAPC) provides a linkage between the cytoskeleton and the extracellular matrix (ECM) and is also a scaffold for a host of signaling molecules. The constituents of the DAPC must be targeted to the sarcolemma in order to properly function. Biglycan is an ECM molecule that associates with the DAPC. Here, we show that biglycan null mice exhibit a mild dystrophic phenotype and display a selective reduction in the localization of -dystrobrevin-1 and -2, - and 1-syntrophin, and nNOS at the sarcolemma. Purified biglycan induces nNOS redistribution to the plasma membrane in cultured muscle cells. Biglycan protein injected into muscle becomes stably associated with the sarcolemma and ECM for at least 2 wk. This injected biglycan restores the sarcolemmal expression of -dystrobrevin-1 and -2, and 1- and 2-syntrophin in biglycan null mice. We conclude that biglycan is important for the maintenance of muscle cell integrity and plays a direct role in regulating the expression and sarcolemmal localization of the intracellular signaling proteins dystrobrevin-1 and -2, - and 1-syntrophin and nNOS.--Mercado, M. L., Amenta, A. R., Hagiwara, H., Rafii, M. S., Lechner, B., Owens, R. T., McQuillan, D. J., Froehner, S. C., Fallon, J. R. Biglycan regulates the expression and sarcolemmal localization of dystrobrevin, syntrophin, and nNOS.