Aggrecan is a major structural component of cartilage extracellular matrix and a specific gene product of differentiated chondrocytes. cDNA clones have been used to isolate rat aggrecan genomic clones from phage and cosmid libraries, producing over 80 kilobases (kb) of overlapping DNA containing the complete rat aggrecan gene, including 12 kb of 5'- and 8 kb of 3'-flanking DNA. DNA sequencing shows 18 exons, most of which encode structural or functional modules; exceptions are domains G1-B and G2-B, which are split into two exons and the G3 lectin domain, which is encoded by three exons. There is one expressed epidermal growth factor-like exon and in addition a non-expressed "pseudo-exon" encoding a heavily mutated epidermal growth factor-like domain. Intron sizes have been determined by restriction mapping and inter-exon polymerase chain reaction; a 30-kb intron separates exons 1 and 2. Exon 1 has been mapped by primer extension and S1 nuclease protection; it encodes 381 base pairs (bp) of 5'-untranslated sequence. There is a minor promoter which initiates transcription an additional 68 bp 5' of the major promoter start site. DNA sequence is reported for a 529-bp fragment encompassing exon 1, including 120 bp of 5'-flanking DNA comprising the promoter. This promoter is lacking the TATAA or CCAAT elements but has several putative binding sites for transcription factors. A 922-bp DNA fragment with 640-bp 5'-flanking DNA and 282-bp exon 1 sequence showed higher promoter activity in transfected chondrocytes than in fibroblasts, is completely inactive in the reverse orientation, and is strongly enhanceable in the forward direction by the SV40 enhancer.