We have isolated and sequenced a Drosophila genomic DNA that encodes the entire coding region of the laminin B1 chain. The genomic DNA sequenced spans 11,787 bp, including a 1.1-kb 5'-flanking region, 5 exons, 4 introns, and a 1.4-kb 3'-flanking region. The open reading frame is within the two largest exons, the exons 3 and 4, while the first two and the last exons are much smaller and are untranslated. The structure of the Drosophila laminin B1 gene is similar to the Drosophila laminin B2 gene. Their exon-intron lengths and Eco RI, Pst I restriction maps are quite conserved. Both of their open reading frames are very compact, and their first introns are much larger than all of the rest of the introns. These results are consistent with the suggestion that the B1 and B2 genes could be derived from an ancestral gene. The similarity of the proximal 5'-flanking regions of the Drosophila B1 and B2 genes is 46.6%. Also, similar sequences of transcriptional regulatory elements, even though not site conserved, are found in both proximal 5'-flanking regions of the B1 and B2 genes. When transfected into Drosophila SL-2 cells, pCAT plasmid containing 1,048 bp of 5'-flanking region shows a strong expression of chloramphenicol acetyltransferase (CAT) activity. The deletion clones that contain sequences between nucleotides -462 to +150, and -282 to +150 all show strong CAT activity. These results suggest that this 5'-flanking promoter region may contain DNA sequences that can promote the expression of the laminin B1 gene.