The allotypes of the C7 M/N polymorphism are determined by comparing the ELISA reaction pattern of the allospecific mAb WU 4-15 with polyclonal anti-C7 IgG. To characterize the molecular basis of this polymorphism, the WU 4-15 epitope was mapped by expression of cDNA fragments. It was found to be located within the boundary region of the two short consensus repeats of C7 (amino acid residues 595-612). Coincidentally an A or C substitution was found in the course of investigation of the gene structure at nucleotide 1759. This leads to an electrically neutral Pro/Thr substitution at residue 565 and alternative Sau961 or RsaI restriction sites, respectively. Enzymatic digestion or sequencing of PCR-amplified genomic DNA from C7 M/N-typed individuals showed a perfect correspondence of the genotype with the phenotype of the C7 M/N polymorphism. In contrast, no association was found with the genotypes of a second polymorphic site at residue 367. Thus, a threonine at amino acid residue 565 allows access of WU 4-15 to its epitope but its substitution with proline almost completely inhibits access in the native molecule, although the epitope is about 40 amino acid residues distant from the polymorphic site in the primary structure. Furthermore, the different conformational structure is probably responsible for the hypomorphic appearance of C7*N.