A precise molecular map of the nidogen binding site of laminins was obtained by site‐directed mutagenesis and structural analysis of the 56 residue LE module gamma1III4 of their gamma1 chain. This demonstrated the crucial importance of the sequence DPNAV (position 800–804) in the disulfide‐bonded loop a, with major contributions made by all residues except P801. Different substitutions of these residues emphasized the essential role of the negative charge (D800) and carboxamide group (N802) as well as their spacings and hydrophobic contacts (V804) for interaction, and predict direct contacts of these three residues with a complementary binding region of nidogen. An inactivating A803‐V substitution, however, may lead to a distorted loop structure. A lower but still significant contribution originates from the non‐contiguous link/loop c sequence LKCIY (positions 815–819) which is spatially close to the loop a sequence. The link residues (L815 and K816) provide main chain hydrogen bonds to N806 and a side chain hydrogen bond to the V804 carbonyl and thus stabilize the conformation of loop a. The side chains of I818 and Y819 together with P842 from loop d form hydrophobic contacts that provide further stability but could possibly also participate in direct ligation. The nidogen binding epitope is therefore localized on a narrow ridge and has a length of approximately 17 angstroms. The data also indicate a strong conservation of the epitope in the laminin gamma1 chains of several invertebrates.