An arginine-specific mono-ADP-ribosyltransferase is expressed on the surface of differentiated mouse skeletal muscle cells and is anchored in the membrane via a glycosylphosphatidylinositol tail. Following incubation of intact cells with [adenylate-32P]NAD and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 97-kDa [32P]ADP-ribosylated protein was observed under reducing conditions and a 140-kDa complex under nonreducing conditions. The ADP-ribosylated protein was purified on a laminin affinity column. Based on its N-terminal sequence (FNLDVM-GAIRKEGEPGSLFGF) and a partial internal sequence (GLMRSEELSFVAGAP), the modified protein was identified as integrin alpha 7. Following partial trypsin digestion, a 39-kDa/79-kDa radiolabeled fragment was produced (reduced/nonreduced SDS-PAGE), narrowing the ADP-ribosylation site to a 39-kDa segment in the extracellular domain of integrin alpha 7. Labeling under optimal conditions was at least 0.4 mol of ADP-ribose/mol of integrin alpha 7. Selective expression of both ADP-ribosyltransferase and integrin alpha 7 in cardiac and skeletal muscle, a similar developmental appearance, and the apparently specific ADP-ribosylation, are consistent with a regulatory association between these proteins. ADP-ribosylation may modulate integrin receptor signaling and could play a significant role in the regulation of muscle cell function by the extracellular matrix.