To better understand the regulation of insulin-like growth factor binding protein-5 (IGFBP-5) expression, we cloned the IGFBP-5 gene from human genomic libraries and identified a region in the 5' flanking sequence which functions as a promoter. The human IGFBP-5 gene is divided into four exons which, primarily due to a first intron of approximately 25 kilobases, span approximately 33 kilobases of DNA. Southern analysis identified a single copy of the IGFBP-5 gene in the haploid human genome, and several independent mapping strategies found this gene tightly linked with, and in opposite transcriptional orientation to, the IGFBP-2 gene at chromosomal region 2q33-34. Primer extension studies identified the IGFBP-5 mRNA cap site 772 base pairs (bp) 5' to the first nucleotide of the translation start codon. Analysis of the 5'-flanking sequence identified a potential TATA element beginning 33 bp 5' to the mRNA cap site. When a DNA fragment containing this cap site and 461 bp of upstream sequence was placed 5' to the chloramphenicol acetyltransferase reporter gene and transfected into MDA-MB-468 human breast cancer cells, it directed chloramphenicol acetyltransferase expression in an orientation-specific manner, suggesting that this region contains elements essential for IGFBP-5 promoter activity.