We have used an expression cloning strategy based on a cell-attachment assay screen to seek identification of molecules required in cellular responses to thrombospondin-1, a regulated macromolecular component of extracellular matrix. We report the identification and functional characterization of a novel, widely expressed, intracellular protein, named muskelin, which contains dispersed motifs with homology to the tandem repeats first identified in the Drosophila kelch ORF1 protein. In adherent C2C12 cells, muskelin localizes in the cytoplasm and at cell margins. Over-expression of muskelin in C2C12 cells promotes cell attachment to the thrombospondin-1 C-terminal domain, alters the mechanisms of attachment to intact thrombospondin-1 and correlates with decreased formation of fascin microspikes and increased assembly of focal contacts by cells adherent on thrombospondin-1. Reciprocally, cell attachment, spreading and cytoskeletal organization are specifically reduced in TSP-1-adherent cells after antisense depletion of muskelin. These results establish a requirement for muskelin in cell responses to thrombospondin-1 and demonstrate that such responses involve a novel process which is integrated into the regulation of cell-adhesive behaviour and cytoskeletal organization.