We isolated and sequenced a Drosophila genomic DNA sequence that encodes the entire coding region of the laminin B2 chain. The 11,464-bp genomic sequence contains a 2.1-kb of 5'-flanking DNA, ten exons, nine introns, and the 3'-flanking region. The first exon encodes a 5' untranslated region; the ATG translational start codon is in exon 2. The entire translated region is within a 8.3-kb Eco RI fragment. The Drosophila laminin B2 gene differs substantially in size and exon pattern from those of the human B1 and B2 genes. However, as in the case of the human B1 gene, the overall exon pattern of the Drosophila B2 gene does not correlate well with the highly conserved structural domains and internal repeats of the B2 polypeptide chain. Unlike the human and mouse B1 and B2 genes, the 2.1-kb 5'-flanking region of the Drosophila B2 gene contains a TATA box and two CAAT boxes. Other potential transcriptional regulatory sequences include two reverse complementary cAMP response element sequences; two sequences that are homologous to the retinoic acid response element motifs of the mouse B1 gene; and sequences homologous to the binding sites for transcription factors dFRA and dJRA, zeste, and possibly GAGA. When transfected into Drosophila SL-2 cells, pCAT plasmid containing 2,090 bp of 5'-flanking region shows a 3.0- to 3.5-fold increase in chloramphenicol acetyltransferase activity after induction with retinoic acid and/or 8-bromo-cAMP. These results suggest that this 5'-flanking promoter region may contain DNA sequences that can regulate the expression of the laminin B2 gene.