The β1D protein is a recently characterized isoform of the integrin β1 subunit that is present in cardiac and skeletal muscles. In this study, we have examined the expression of β1D in different types of skeletal muscle and in cardiac muscle and studied its distribution during mouse development, using new monoclonal antibodies specific for β1D. Immunoprecipitation studies revealed that, while β1A is strongly expressed in proliferating C2C12 myoblasts, β1D is only expressed after their differentiation to myotubes. In these myotubes, β1D is associated with different α subunits, namely α3A, α5, α7A, or α7B. Initially, during embryogenesis, the β1A subunit is the only β1 variant expressed in skeletal and cardiac muscle. The β1D subunit is first detected in skeletal muscle at E17.5, whereas in cardiac muscle its expression begins around the time of birth. Later the expression of β1A in skeletal and cardiac muscle becomes restricted to capillary cells, whereas β1D eventually becomes the only variant expressed in adult cardiac and skeletal muscle cells. The switch from the β1A to the β1D subunit in cardiac muscle cells coincides with the expression of α7. In adults there is a distinct concentration of β1D at the myotendinous junctions of muscle fibers and at costameres in both cardiac and skeletal muscle. In addition, β1D is present at intercalated discs in cardiac muscle and at neuromuscular junctions in skeletal muscle cells. The amount of β1D in different types of skeletal muscle (fast, slow, and mixed‐type) was similar, but cardiac muscle expressed almost five times as much of this protein. We suggest that β1D plays a role in the maintenance of the cytoarchitecture of mature muscle and in the functional integrity of the muscle cells. Dev. Dyn. 1997;210:472–486. © 1997 Wiley‐Liss, Inc.