Expression of the laminin-binding alpha7 integrin is tightly regulated during myogenic differentiation, reflecting required functions that range from cell motility to formation of stable myotendinous junctions. However, the exact mechanism controlling alpha7 expression in a tissue- and differentiation-specific manner is poorly understood. This report provides evidence that alpha7 gene expression during muscle differentiation is regulated by the c-Myc transcription factor. In myoblasts, alpha7 is expressed at basal levels, but following conversion to myotubes the expression of the integrin is strongly elevated. The increased alpha7 mRNA and protein levels following myogenic differentiation are inversely correlated with c-Myc expression. Transfection of myoblasts with the c-Myc transcription factor down-regulated alpha7 expression, whereas overexpression of Madmyc, a dominant-negative c-Myc chimera, induced elevated alpha7 expression. Functional analysis with site-specific deletions identified a specific double E-box sequence in the upstream promoter region (-2.0 to -2.6 kb) that is responsible for c-Myc-induced suppression of alpha7 expression. DNA-protein binding assays and supershift analysis revealed that c-Myc forms a complex with this double E-box sequence. Our results suggest that the interaction of c-Myc with this promoter region is an important regulatory element controlling alpha7 integrin expression during muscle development and myotendinous junction formation.