The rat bone Gla protein (BGP, osteocalcin) gene was isolated from a rat genomic library and sequenced. BGP is a 5.8-kD noncollagenous protein secreted by calcified tissues whose expression is regulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The BGP gene consists of 4 exons separated by 3 introns and spans almost 1 kb of DNA. The 5'-flanking region of the gene contains CCAAT and TATAAA elements common to eukaryotic genes. An analysis of approximately 600 bp of 5'-flanking sequence of this gene revealed sequences homologous to regulatory elements for glucocorticoids, metal ions, and cAMP. The latter is especially significant since recent evidence suggests that the rat BGP gene can be regulated by cAMP. This region of the gene also contains numerous pairs of inverted repeat sequences (imperfect palindromes). The sequence of the rat BGP gene was compared to that of the recently published human BGP gene (Celeste et al., EMBO J. 5, 1885, 1986). The coding regions of these two genes share 77% sequence identity, and several regions of the 5'-flanking sequences are also well conserved. Knowledge of the sequence of the rat BGP gene will allow studies of its regulation by 1,25(OH)2D3, cAMP, and other trans-acting transcriptional factors, and identification of the regulatory sequence elements involved.