We use immunoblotting, immunoprecipitation, and centrifugation in sucrose density gradients to show that the product of the erythrocyte β-spectrin gene in rat skeletal muscle (muscle β-spectrin) is present in two states, one associated with fodrin, and another that is not associated with any identifiable spectrin or fodrin subunit. Immunofluorescence studies indicate that a significant amount of β-spectrin without α-fodrin is present in the myoplasm of some muscle fibers, and, more strikingly, at distinct regions of the sarcolemma. These results suggest that α-fodrin and muscle β-spectrin associate in muscle in situ, but that some muscle β-spectrin without a paired α-subunit forms distinct domains at the sarcolemma. Cell Motil. Cytoskeleton 37:7–19, 1997. © 1997 Wiley-Liss, Inc.