Abstract: High affinity binding of nidogen to laminin is mediated by an EGF-like repeat .gamma.1III4 of the mouse laminin .gamma.1 chain and has now been restricted to two short non-contiguous regions of its 56 residue sequence by use of synthetic peptides and recombinant mutants. Disulfide loop a,b of the repeat and a modified loop a,c could completely inhibit binding, with a 5,000-fold or 300-fold reduced affinity, respectively. Synthetic loops c and d lacked inhibitory activity. Some binding contribution of Try819 in loop c was, however, shown by mutation and side chain modification. Together with studies of loop chimeras, this indicated a distinct cooperativity between the two binding sites. The major binding site of loop a was localized to the heptapeptide NIDPNAV (position 798-804). A change of Asp800 to Asn or Ala803 to Val caused a strong reduction in binding activity, while only small effects were observed for the changes Pro801 to Gln and Ile799 to Val.