Nidogen/entactin is a ubiquitous 150-kDa multidomain basement membrane protein. Since in vitro binding studies indicated that nidogen may function as a major mediator in basement membrane organization and assembly, analysis of gene structure and regulation of gene expression will help us to understand many biological processes that involve degradation and reorganization of the basement membrane zone. An approximately 100-kb region of genomic DNA encoding the human nidogen gene (NID) including 5' and 3' flanking sequences has been cloned and characterized by restriction mapping and sequencing. The entire gene is more than 90 kb in length and contains 20 exons. All introns interrupt protein coding sequences. The size of individual introns varies significantly, ranging from 0.6 to 18 kb. Its exon/intron structure revealed that the protein domains of human nidogen are organized in a domain-specific manner with various subdomains being encoded by individual exons, indicating that exon duplication and shuffling have played an important role in determining the present structure of the protein. Comparison of the exon organization with the recently published ascidian nidogen amino acid sequence strongly suggests that vertebrate nidogen might have evolved from a common ancestral precursor resembling ascidian nidogen.