Dystroglycan (DG) is an extracellular receptor composed of two subunits, a-DG and b-DG, connected through the a-DG C-terminal domain and the b-DG N-terminal domain. We report an alanine scanning of all DG cysteine residues performed on DG-GFP constructs overexpressed in 293-Ebna cells, demonstrating that Cys-669 and Cys-713, both located within the b-DG N-terminal domain, are key residues for the DG precursor cleavage and trafficking, but not for the interaction between the two DG subunits. In addition, we have used immunprecipitation and confocal microscopy showing that ERp57, a member of the disulfide isomerase family involved in glycoprotein folding, is associated and colocalizes immunohistochemically with b-DG in the ER and at the plasma membrane of 293-Ebna cells. The b-DG-ERp57 complex also included a-DG. DG mutants, unable to undergo the precursor cleavage, were still associated to ERp57. b-DG and ERp57 was co-immunoprecipitated also in rat heart and kidney tissues. In vitro, a mutant ERp57, mimicking the reduced form of the wild-type protein, interacts directly with the recombinant N-terminal domain of both a-DG and b-DG with apparent dissociation constant values in the micromolar range. ERp57 is likely to be involved in the DG processing/maturation pathway, but its association to the mature DG complex might also suggest some further functional role that needs to be investigated.