Bone sialoprotein (BSP) is a major structural protein of the bone matrix that is specifically expressed by fully-differentiated osteoblasts. To characterize the gene and to study the tissue-and differentiation stage-specific regulation of BSP gene transcription we have isolated and partially sequenced two overlapping genomic fragments which span the complete human BSP gene and its promoter region. The approximately 15 kb gene comprises seven exons of 82 bp, 68 bp, 51 bp, 78 bp, 63 bp, 159 bp and 2.5 kb (1-7, respectively), separated by six introns of approximately 3 kb, 92 bp, 95 bp, approximately 3 kb, approximately 0.5 kb and approximately 4.5 kb. All of the intron-exon boundaries defining the splice sites conform to the consensus sequence of: AG at the 3' splice site; and GT at the 5' splice site, except the 3' splice site of exon 1. The first exon encodes the 5'-UTR, the second exon the signal sequence and the first two amino acids, exons 3 and 4 the Tyr-and Phe-rich amino terminus, and exon 5 the first segment of polyglutamic acid. Exon 7 encodes over half of the protein including a second polyglutamic acid segment, the RGD cell attachment motif, the sulphated tyrosine-rich C-terminus and the 3'-UTR. The promoter region is characterized by an inverted TATA-like sequence (TTTATA), nts -28 to -23 from the transcriptional start site (+1), and an inverted CCAAT box (ATTGG) at -54 to -50. Analysis of chimeric constructs fused to a CAT reporter gene indicate that the presence of both the inverted TATA-like sequence and CCAAT elements are required for basal promoter activity. Comparison of the human BSP promoter with the rat BSP promoter (Li and Sodek, 1993) reveal that the nature and position of the inverted TATA-like sequence and CCAAT box together with an Ap1 (-148 to -142), CRE (-122 to -116) and a homeobox-binding site (-200 to -191) have been conserved. A putative Glucocorticoid Response Unit (GRU) consisting of a Glucocorticoid Response Element (GRE) and an overlapping direct repeat (DR5) similar to the retinoic acid receptor element (RARE) is present at -1038 to -1022. These studies have defined the structure of the human BSP gene and have identified novel transcriptional elements in the promoter that may be involved in the developmentally regulated, tissue specific expression of this gene.