The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 ± 0.005 mol/mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent K(d) for dystrophin of 0.5 μM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking superfamily of proteins. However, we failed to observe dystrophin-glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with α-actinin. Furthermore, high speed cosedimentation analysis of dystrophin-glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F-actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side- binding proteins.