The binding specificity of alpha7beta1 integrins for different laminin isoforms is defined by the X1 and X2 splice domains located in the beta-propeller domain of the alpha7 subunit. In order to gain insight into the mechanism of specific laminin-integrin interactions, we defined laminin-binding epitopes of the alpha7X1 and -X2 domains by single amino acid substitutions and domain swapping between X1 and X2. The interaction of mutated, recombinantly prepared alpha7X1beta1 and alpha7X2beta1 heterodimers with various laminin isoforms was studied by surface plasmon resonance and solid phase binding assays. The data show that distinct clusters of surface-exposed acidic residues located in different positions of the X1 and the X2 loops are responsible for the specific recognition of laminins. These residues are conserved between the respective X1 or X2 splice domains of the alpha7 chains of different species, some also in the corresponding X1/X2 splice domains of alpha6 integrin. Interestingly, ligand binding was also modulated by mutating surface-exposed hydrophobic residues (alpha7X1L205, alpha7X2Y208) at positions corresponding to the fibronectin binding synergy site in alpha5beta1 integrin. Mutations in X1 that affected binding to laminin-1 also affected binding to laminin-8 and -10, but not to the same extent, thus allowing conclusions on the specific role of individual surface epitopes in the selective recognition of laminin-1 versus laminins -8 and -10. The role of the identified epitopes was confirmed by molecular dynamics simulations of wild-type integrins and several inactivating mutations. The analysis of laminin isoform interactions with various X1/X2 chimaera lend further support to the key role of negative surface charges and pointed to an essential contribution of the N-terminal TARVEL sequence of the X1 domain for recognition of laminin-8 and -10. In conclusion, specific surface epitopes containing charged and hydrophobic residues are essential for ligand binding and define specific interactions with laminin isoforms.