Fibulin-2, an extracellular matrix protein containing tandem arrays of calcium-binding epidermal growth factor-like motifs, is present in the basement membrane and stroma of many tissues. Its expression pattern suggested an essential role in organogenesis, particularly in embryonic heart development. In this study, we cloned the extreme 5' end of the mouse fibulin-2 cDNA, isolated phage and cosmid clones encoding the entire gene, and functionally characterized the promoter. The gene was found to consist of 18 exons spanning 55 kb of DNA. The exon-intron organization reflected the modular structure of the protein. Exon 9 was subjected to alternative splicing. All splice junctions conformed to the GT/AG rule, except that GC instead of GT was found in the splice donor site of exon 4. The gene lacked TATA and CAAT boxes but contained an initiator element (Inr) and several consensus Sp1 binding sites surrounding the transcription start sites. By transient transfection of promoter deletion constructs, a 0.46-kb region containing the clustered Sp1 sites was found to confer a high promoter activity.