The complete intron-exon organization of the gene encoding human perlecan (HSPG2), the major heparan sulfate proteoglycan of basement membranes, has been elucidated, and specific exons have been assigned to coding sequences for the modular domains of the protein core. The gene was composed of 94 exons, spanning > 120 kbp of genomic DNA. The exon arrangement was analyzed vis-à-vis the modular structure of the perlecan, which harbors protein domains homologous to the low density lipoprotein receptor, laminin, epidermal growth factor, and neural cell adhesion molecule. The exon size and the intron phases were highly conserved when compared to the corresponding domains of the homologous genes, suggesting that most of this modular proteoglycan has evolved from a common ancestor by gene duplication or exon shuffling. The 5' flanking region revealed a structural organization characteristic of housekeeping and growth control-related genes. It lacked canonical TATA or CAAT boxes, but it contained several GC boxes with binding sites for the transcription factors SP1 and ETF. Consistent with the lack of a TATA element, the perlecan gene contained multiple transcription initiation sites distributed over 80 bp of genomic DNA. These results offer insights into the evolution of this chimeric molecule and provide the molecular basis for understanding the transcriptional control of this important gene.