The exon-intron structure of the human laminin B1 chain gene was determined from genomic clones that spanned 90 kilobase pairs (kb), including 80 kb of the structural gene, about 1 kb of the 5'-flanking region, and 9 kb of the 3'-flanking region. DNA sequencing and heteroduplex analyses demonstrated that the gene consists of 34 exons. The intron sizes vary from 92 base pairs to more than 15,000 base pairs. The clones did not completely contain introns 13 and 14 and, therefore, the exact size of the gene remains to be determined. The first exon encodes a 5'-untranslated region, with the ATG translation start codon being in exon 2. The promoter region does not contain TATA or CAAT boxes, but it has four GC boxes. Additionally, two GC boxes are located in the 5'-untranslated sequence. A glucocorticoid response element-like sequence and a sequence resembling the binding sequence for transcription factor AP-2 are present in the 5'-flanking region. Another potential AP-2 binding sequence is located in the 5'-untranslated sequence of exon 1. A TGACC motif present in the binding region for the retinoic acid response element of the mouse laminin B1 gene promoter is also present in the human counterpart. The overall exon pattern of the gene correlates only slightly with the highly conserved structural domains and internal repeats of the B1 polypeptide chain. Furthermore, the exon profile differs considerably from that of the laminin B2 gene. A HineII/HpaI restriction fragment length polymorphism was identified in exon 31.